Arthrospira (Spirulina) 2 5 Claudio Sili , Giuseppe Torzillo and Avigad Vonshak

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

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

B.A. Whitton (ed.), Ecology of Cyanobacteria II: Their Diversity in Space and Time, 677 DOI 10.1007/978-94-007-3855-3_25, © Springer Science+Business Media B.V. 2012 678 C. Sili et al. genus Spirulina Turpin 1829 . Most authors followed Geitler for a long while, but increasingly researchers realized that 25.2 Morphology genera are distinct and returned to using two names. The evi- dence supporting this was summarized by Castenholz ( 2001 ) The main morphological feature of Arthrospira is the typical and Komárek and Anagnostidis (2005 ) . Many species cur- arrangement of its multicellular cylindrical trichomes in an rently listed as Spirulina should therefore be re-included in open helix usually of relatively large diameter, sometimes Arthrospira and these include all those grown commercially attenuated at the ends, and with evident cross-walls and sold as Spirulina. This material is now known so widely (Fig. 25.1). In contrast, Spirulina presents a screw-like under this name that it seems inevitable that the name will trichome, generally with almost closed, uniform and narrow persist; however, it should be written as Spirulina or spirulina diameter screws (0.5–3 mm), cells with cross-walls usually i.e. no italics. invisible at light microscope, without gas vacuoles and with Arthrospira has been reported to exist in environments prominent granules (Fig. 25.2 ). The trichomes of Arthrospira varying in their osmoticum, temperature and salt concentra- are composed of cylindrical cells that undergo binary fi ssion tions, most being of high alkalinity (Iltis 1969a, b ; Busson in a single plane perpendicular to the main axis. Trichome 1971 ) . Filaments of (true) Spirulina also occur in many of elongation occurs through multiple intercalary cell division these environments, but apparently never forming the blooms along the entire fi lament. Multiplication occurs only by frag- that often occur with Arthrospira . mentation of a trichome, usually in correspondence of a There is great interest in past and present use of Arthrospira necridial cell (Fig. 25.3 ). The mechanism, has been described as a food, though nowadays mostly as a “health” food. in detail for both A. maxima and A. fusiformis by Tomaselli Dangeard (1940 ) , Brandily ( 1959 ) and Léonard and Compère et al. ( 1981 ) . It consists in the destruction of an intercalary (1967 ) all described how African tribes living along Lake sacri fi cial cell (necridium) that fi rst becomes colourless and Chad collect this alga. The biomass is harvested from water- fi nally biconcave due to the collapse of the lateral septa. bodies near the lake and sun-dried on the shores to produce a However, the presence of necridia become less evident when hardened dark cake called “dihé”, which is broken into small cultures are subject to fast mixing as it occurs in mass cul- pieces and used in different forms by the local populations as tures. In contrast, the fragmentation of Spirulina trichomes part of their daily diet. At about the same time, Arthrospira occurs always without the production of necridic cells was also recorded in the water of Lake Texcoco, Mexico. (Komárek and Anagnostidis 2005 ) (Table 25.2 ) . Here, it had been used as food by the natives living in The trichome width of Arthrospira populations sampled the area (Clément 1968 ) . Travellers to Mexico during the from nature ranges from about 2.5 to 16 m m, while the helix sixteenth century described how the Aztecs used a soft a pitch typically ranges from 0 to 80 m m and its diameter from blue-green material, harvested with fi ne nets from the lake, 15 to 60 m m. The dimensions and other morphological fea- for making a kind of bread called “tecuitlatl” (Ciferri 1983 ) . tures of A . fusiformis (i.e. the well-known commercial It is striking that these two human populations, living far Spirulina platensis) not only vary markedly between popula- apart, discovered the nutritional value of Arthrospira tions, but also within one population (Fig. 25.4). Both under independently. laboratory and mass cultivation conditions, the helix architec- Later, attention was refocused on “Spirulina” by the pio- ture (pitch and diameter) is highly dependent on growth and neering work of the Institute Française du Pétrole on environmental conditions. Observations on Arthrospira cyanobacterial blooms in the evaporation ponds of the indus- fusiformis by the authors have shown that morphological trial soda production facility at Lake Texcoco near Mexico variability and trichome motility are especially evident during City. This led to the fi rst detailed study of the growth require- the fi rst weeks following trichome isolation, and that this is ments and physiology of Arthrospira . The Ph.D. research of usually maintained for some years in laboratory liquid culture Zarrouk (1966 ) was the basis for establishing the fi rst large- (Fig. 25.5 ). In contrast, morphological variation in A. maxima scale production plant. The work was followed up by several following its isolation is much less marked (Fig. 25.6 ). Both groups in Italy, France and Israel and summarized in a review A. fusiformis (Fig. 25.7 ) and A. maxima typically have abun- by Ciferri ( 1983 ) . The subsequent extensive research on cell dant gas vacuoles, which help to position the organism in the biology, biochemistry and biotechnology has been reviewed water column. They have an important role in the harvesting in books edited by Vonshak ( 1997a ) , and Richmond ( 2004a ) . of the bloom by African people who utilize A. fusiformis for This chapter provides a perspective on the biology and bio- the preparation of dihè (Abdulqader et al. 2000 ) . technology of the two most important species of Arthrospira The sheath of Arthrospira is usually absent in planktonic utilized for commercial mass cultivation, A. maxima and A. populations. Where a sheath is present, it is colourless, tube- fusiformis (usually reported as Spirulina maxima and like, adjacent to the trichome, open at the ends and contains Spirulina platensis, respectively). only one trichome (Komárek and Hauer 2011 ) . In contrast, a 25 Arthrospira (Spirulina) 679

Fig. 25.1 Morphological aspects of the evident cross-walls in formaldehyde (2%); (c ) fresh clonal trichome grown in mass culture; Arthrospira fusiformis isolated from soda Lake Kailala (Chad). ( a ) (d ) auto fl orescence of trichome shown in (c ). Bar marker = 10 m m Fresh clonal trichome grown in laboratory; (b ) wild trichome fi xed in (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)

considerable production of mucilage can be formed by a capsular investment, start to increase (Fig. 25.8a ). In some strain of A. fusiformis, which was isolated from Lake Kailala, cases, it is possible to observe a single wide mucilaginous on agar plates, particularly if the material starts to dry out envelope surrounding each trichome (Fig. 25.8b ). The rupture (authors, unpublished data). Under such conditions, cocoon-like of the capsule causes the release of 2–4 trichomes (Fig. 25.8c ). shapes, which are generally immersed in a polysaccharidic The polysaccharide slime produced by A. fusiformis on agar matrix, with two or more entangled trichomes inside their plates can be dense (Fig. 25.8d ). It seems likely that these 680 C. Sili et al.

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) structures are an adaptation to cope with adverse environ- Further studies revealed that under growth conditions, the mental conditions, such as those in the semi-dry zones at the linear trichomes could spontaneously revert to the helical edge of the small alkaline lakes with A. fusiformis blooms, trichomes with the same morphology as the original ones. which are often subject to rapid and extensive draining with These authors also observed, there were signi fi cant differ- the onset of the dry season. ences in morphology, ultrastructure, physiology, biochemis- The effect of temperature on fi lament structure was stud- try, and genetic characteristics between the linear trichomes ied by Van Eykelenburg (1979 ) , who subsequently described and the original or reverted ones, but no differences were the rapid reversible change from the helix to the spiral shape found between the original (helical) and the reverted on solid media (Van Eykelenburg and Fuchs 1980 ) . Although, trichome, indicating that both linearization and reversion the the simultaneous presence of straight and helicoidal forms of helical shape of Arthrospira trichomes were due to genetical A. fusiformis and A. maxima (Fig. 25.9) has been observed variation. Occasional helical trichomes in cultures of frequently in the laboratory and in mass cultures, the trigger Arthrospira fusiformis strain D885/S which is a straight factor and the mechanism for this morphological change still form, have been also observed by Mühling et al. (2006 ) . remain obscure. A detailed study of the effect of physical However, in all cases the helical trichomes were outcom- and chemical conditions on the helix geometry of Arthrospira peted by straight ones and disappeared after subsequent sub- was done by Jeeji Bai and Seshadri (1980 ) and Jeeji Bai culturing. In contrast, Noor et al. ( 2008 ) found that a stock (1985 ) . They reported the occurrence of the straight or nearly culture of Arthrospira (Spirulina) maintained it helical struc- straight spontaneous culture variants also observed by Lewin ture after 17 years of subculture, while fi laments in pilot ( 1980) . However, there are considerable differences in the ponds lost their helical structure. They concluded that the degree of coiling between different strains of the same spe- prolonged period of acclimation to adverse climatic condi- cies and within the same strain, the helical shape of the tions outdoors, may cause reversion of coiled fi laments to trichome is regarded as a peculiar property of the genus. straight ones, and that this fact supports the idea that straight Once a strain has converted to the straight form, either natu- fi laments may have a higher survival capacity. However, bio- rally or due to physical or chemical treatments, it does not chemical composition and nutritional properties were not usually revert back to the helical form. Jeeji Bai ( 1985 ) sug- affected by the shape of the fi laments (Noor et al. 2008 ; gested that this may be due to a mutation affecting some G. Torzillo, unpublished). trichomes under certain growth conditions. The common Interestingly, Mussagy et al. (2006 ) , starting from a culture occurrence of straight trichomes in mass cultures of of hormogonia of the straight trichomes, obtained one with a Arthrospira also suggests that the helical character may be mix of loosely-coiled and straight ones. Finally, Hongsthong carried on plasmids, but no one has yet demonstrated the et al. (2007 ) , using a proteomic approach, concluded that the existence of plasmids in Arthrospira . It has been reported linear morphology observed both in the laboratory and in that helical shape is related to protection of the organism industrial production ponds was possibly induced fi rstly by against photolysis (Fox 1996 ) . Wang and Zhao ( 2005 ) environmental stresses, such as oxygen level, carbon dioxide observed that the helical fi laments originated from the linear level, nutrient availability and light, and secondly by the fi laments 9 years old obtained with ionizing radiation. change in a cell shape determination mechanism. This study 25 Arthrospira (Spirulina) 681 and capitate outer cell wall outer cell wall Morphology of trichome end + Rounded − Rounded or fl at calyptra + + Conical calyptra + Slightly attenuated end Rounded, no calyptra + Rounded with thickend Gas vacuoles intensely narrowed or intensely narrowed ends widened towards coils +/− densely or loosely coil − Rounded, no calyptra ends towards narrowed coils ends not attenuated towards coiled, gradually slightly attenuated at ends Helix shape ) 2011 20–40 (dense) 5.5–5.8 (loose) m) ) and Komárek and Hauer ( ( and Hauer and Komárek ) m 2005 15–50 0–80 Regularly screw-like coiled (20?) 26–36 (24?) 30–57 8.8–10.8 (dense) loosely spiral +/− regularly 16–39 16–66 Spirally coiled and distinctly Coils ( Width Width Height m) m than wide isodiametric as wide 0.85–1.7 51–6.8 10.2–11.5 Regularly loosely spiral coils − Rounded

m) Cell length ( m (3.4)5–9 (11?) 2–3 × shorter (3.7) 4–6 (8) 5–6 (4) 6–7 (9?) Nearly 2.8–3.8 (2.7) (7.4) 8–15 (17) 3.1–4.3 About 2 +/− (9.2) 12–25 (31) isodiametric 3.1–9.3 coils screw-like Regularly 12.5–18.5 (loose) 9–10.5 − 2–3 7.4–11.8 Rounded, no calyptra 8–9 7–13 20–35 3–5.2 4.2–5.1 1/3–1/2 as 33–49 long Regularly screw-like coil 2.7–3.4 − Loosely screw-like 63–75 6.8–7.6 Rounded 20–22 − 17–19.2 Rounded 20–35 conical Regularly screw-like coils, Regularly loosely screw-like − + Trichome Trichome width ( (6.5) 7–9 (10) 5–7 40–60 70–80 Regularly loosely screw-like 3.5–4 (5) (2.5) 3–4 0.8–1.2 2.5–3 6–7 +/− isodiametric 6–6.5 (8) 11–16 12–15 15–19 7–12 (15) Regularly coiled − − +/− conical − Rounded. no calyptra

species based on Komárek and Anagnostidis ( ( and Anagnostidis species based on Komárek

1892 1992 1892 Arthrospira

Vasishta 1962 Vasishta 1990

Kamat 1963 Setchell 1895 Komárek et Komárek (Voronichin) Wang 1933 Wang (Frenguelli) Kufferath 1914 Kufferath (Nordstedt) Gomont Morphology of Drouet et Stirickland 1917 (Schmidle) Anagnostidis 1998 Setchell et Gardner Stizenberger ex Gomont ex Stizenberger Magrin, Senna et Komárek 1998 Magrin, Senna et Komárek Desikachary et Jeeji Bai Brühl et Biswas 1922 Brühl et Biswas Vasishta 1963 Vasishta A. indica A. massartii A. pellucidis A. platensis A. santannae 2006 Komárková-Legnerová A. skujae A. tenuis (planktonic, solitary trichomes) with gas vacuoles Types A. argentina Guarrera et Kühnemann 1949 A. fusiformis et Lund Komárek A. ghannae in Drouet 1942 A. joshii A. maxima in Gardner Table 25.1 Species Types without gas-vacuoles (forming mats) (forming without gas-vacuoles Types A. balkrishnanii A. desikacharyensis A gigantea A gomontiana A. jenneri 682 C. Sili et al.

Fig. 25.4 Morphological variability in Arthrospira fusiformis in two formaldehyde (2%) from Lake Kossorom. (f ) Shows that occasional ± soda lakes at the irregular north-east fringe of Lake Chad. ( a , b , c ) Wild straight trichomes may also be present in a wild sample. Bar fresh trichomes from Lake Kailala; (d , e , f ) wild trichomes fi xed in marker = 40 m m (Photos C. Sili) was the fi rst to show evidence at the protein level that could arti fi cial laboratory conditions favour the development of explain this morphological transformation in Arthrospira . straight forms. After 8 years of repeated transplants in liquid The effect of solar UV radiation on morphology on culture, two A. fusiformis strains (Kailala and Kossorom) Arthrospira was studied by Wu et al. (2005 ) using an indoor had about 30% had straight trichomes (Fig. 25.10a ), 50% grown strain, which had not been exposed to sunlight for with loosely-coiled trichomes (Fig. 25.10b ), while only 20% decades, and an outdoor-grown strain grown under sunlight maintained their original or an elevated degree of helicity for decades. The experiments con fi rmed what had already (Fig. 25.10c ) (C. Sili, unpublished data). observed by Jeeji Bai and Seshadri (1980 ) that the self-shad- A survey of the morphological characters of 36 clonal ing effect produced by compression of the spirals over adap- axenic strains of cultivated Arthrospira carried out by tive time scales seem to play an important role in protecting Mühling et al. (2003 ) showed that of the 34 with helical this species against deleterious UV radiation. Gao et al. trichomes, fi ve were right-handed and 29 left-handed. After (2008 ) described interactions between temperature and UVR 1 year of repeated subculture, the orientation of one helical on the morphology of A. fusiformis and found that UVR strain had changed from right to left-handed, suggesting a caused a breakage of the spiral structure at 15°C and 22°C, probable genetic shift. In addition, a temperature shift from but not at 30°C. A. fusiformis was able to modify its spiral 30°C to 34°C for 7 days led to a change in orientation in three structure by increasing helix tightness at the highest temper- strains, which was reversible if the temperature shift was ature (30°C). PAR has been found to cause a decrease in the reversed. The fact that the orientation of trichome helix can be helix pitch, while UVR increased the extent to which helices reversed by genetic drift and by environmental factors such are compressed (Ma and Gao 2009 ) . In general it seems that as temperature upshift and mechanical stress demonstrates

Fig. 25.5 Morphological variability in fresh clonal trichomes of Arthrospira fusiformis isolated from soda Lake Kailala (Chad) and grown in the laboratory. Bar marker = 20 m m (Photos C. Sili)

Fig. 25.6 ( a) Morphological aspects of regular clonal trichomes of cells diameter at the ends. Bar markers correspond to 40 m m in a and Arthrospira maxima isolated from the solar Lake Texoco evaporator. 20 m m in b (Photos C. Sili) (b ) Typical trichome regularly screw-like coiled with gradually attenuated

Fig. 25.7 ( a ) Clonal trichomes of Arthrospira fusiformis isolated from the soda Lake Kailala (Chad) with a typical spiral markedly narrowed towards the ends. (b ) Dark- 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 typical cocoon-like shaped after their release as single fi laments; Petri dishes. (a ) Cocoon-like shape immersed in a polysaccharide (d ) cocoon-like shaped and free trichomes immersed in a dense slime matrix, with two or more internal entangled motile trichomes; ( b ) of polysaccharides negatively stained with Indian ink. Bar markers two single trichomes surrounded by mucilaginous envelopes that, in correspond to 40 m m in ( a ) and (c ), 20 m m in (b ) and 100 m m in turn, are surrounded by thick capsules; (c ) clusters of trichomes of a (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) 25 Arthrospira (Spirulina) 685

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 stress; arrow indicates the position on the trichome where the helix Arthrospira fusiformis cultures. (A ) left-handed helices, (a ); right-handed orientation reversed from left-hand (a ) to right-hand (b ). Bar marker = 20 m m helices (b ). ( B) Example of reversal helix orientation due to mechanical (Photos C. Sili)

that care is needed in using this character for taxonomic pur- slight progressive increase in mean sinking speed with poses (Mühling et al. 2003 ) . Occasionally, reversed forms increasing length helical fi laments though up to 10–20 cells. from left-to- right handed orientation have been observed in The authors concluded the advantage of straight fi laments our Arthrospira fusiformis laboratory and mass cultures could not be in conferring a higher growth rate, but it might (Fig. 25.11 ). be in resisting predation or affecting the rate of sinking and Sinking velocity is markedly infl uenced by the shape of fl oating. Similar conclusions were drawn by Mühling et al. trichomes. If a planktonic species evolves towards a decreas- ( 2003) . These authors reported that in one example of graz- ing sinking velocity, it has three options: (i) it may decrease ing by large ciliate, the ciliate had to make a rotatory motion its body size (but this could increase the risk of being grazed almost following the coil of the Arthrospira , with up to 10 by zooplankton); (ii) it may decrease its specifi c gravity (e.g. coils showing inside the ciliate. It was also suggested that, increasing gas vacuole density) or reducing the amount of perhaps the reversal of helix orientation at intervals within a ballast substances (e.g. carbohydrate); (iii) or may increase particular ecosystem in response to shifts between grazers or its form resistance. Padisák et al. ( 2003 ) investigated the changes within populations of one particular grazer. sinking velocities of different PVC models of phytoplankton The ultrastructural cell organization of Arthrospira is typ- organisms. The sinking velocity was related to dimension- ical of prokaryotes, with a lack of membrane-bound organ- less form resistance factor (F ). It was observed that F elles (Van Eykelenburg 1979 ; Vonshak and Tomaselli 2000 ) . decreased in coiled shaped organisms, and increased with The thin cell wall has four layers, with an easily detectable the straight ones. As a result coiled forms tend to sink at a electron-dense layer corresponding to the peptidoglycan faster speed than straight ones. These fi ndings agree with layer (Van Eykelenburg 1977 ) . The regularly spaced cross- those made by Booker and Walsby (1979 ) on Anabaena fl os- walls divide the trichome into cells connected by plasmodes- aquae . They observed that helical fi laments of all length mata. Cross-walls are formed by centripetal growth and classes sank signi fi cantly faster than straight ones of corre- extensions of both the peptidoglycan and the more internal sponding length. In the case of straight fi laments there was a layer of the cell wall toward the centre of the cell. The cell 686 C. Sili et al. has a number of inclusions mostly corresponding to the was considered to be unicellular. Some years later, typical ones for cyanobacteria described in previous books Stizenberger (1852 ) , who had observed septa in some heli- (Fogg et al. 1973; Carr and Whitton 1973, 1982 ) . Thylakoid cally coiled oscillatoriacean forms, proposed that the forms, membranes with phycobilisomes are arranged in parallel with a multicellular structure, be included in a new genus, bundles. The other main subcellular inclusions are (in addi- Arthrospira. This distinction was confi rmed later by Gomont tion to carboxysomes, ribosomes and DNA fi brils) gas vacu- ( 1892 ) in his taxonomic study on the Oscillatoriaceae. He oles, polyglucan granules, polyphosphate granules and large left the apparently aseptate forms ( trichomata unicellularia ) cyanophycin granules. The extent of development of the last in Spirulina and placed the septate forms ( trichomata pluri- three depends on the nutrient status of the trichome; for cellularia ) in Arthrospira Stizenberger 1852 . instance N-rich conditions favour cyanophycin formation. However, Geitler’s revision (1925 , 1932) of the Cyanop- Structures like the cylindrical bodies reported for some hyceae re-uni fi ed the members of these two genera in other members of the Oscillatoriaceae are often present. Spirulina Turpin 1829. He based classifi cation on the close These inclusions, whose explanation is still unknown, are similarity in morphology and ignored the presence of septa lacking in Spirulina (Tomaselli et al. 1996 ; Tomaselli 1997 ) . and thus made no distinction between the forms previously Studies carried out by Palinska and Krumbein ( 2000 ) on the recognized as separate genera. The genus was thus divided peptidoglycan cell wall of cyanobacteria confi rmed the by Geitler into two subgeneric taxa (Section I. Arthrospira observations made by Guglielmi and Cohen-Bazire ( 1982 ) and Section II. Euspirulina) on the basis of the criterion orig- concerning different perforation patterns of Spirulina and inally used by Stizenberger (1852 ) to separate the genera Arthrospira, which were used as a criterion in order to sepa- Spirulina and Arthrospira i.e. visible or non-visible cross- rate them clearly into two different genera. Spiller et al. walls. The forms with septa that were easily observable under (2000 ) , using an x-ray microscope, showed that the cell end a light microscope were classi fi ed in Section Arthrospira and walls were clearly visible in A. fusiformis , and that they those with unlikely or only artifi cially observable septa crossed the fi lament at intervals of about 2–3 m m. The images (after trypsin treatment or staining with neutral red) in also revealed two interesting structures characterised by a Section Euspirulina. spherical shape, as well as a looping strand that resembled In accordance with those authors who sustain that many beads on a string. 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 25.3 Taxonomy genera: SpirulinaTurpin with invisible or unicellular cells of trichome, and Arthrospira Stizenberger with cells of the As discussed in the introduction section, the question of the trichome clearly visible. Subsequently, Bourrelly (1970 ) , by taxonomy and nomenclature of Arthrospira and Spirulina following a radical taxonomy attribution, merged Spirulina was debated for almost the entire twentieth century. Several and Arthrospira in Oscillatoria, and remarked that new questions have been solved only in recent decades with the understanding of cell colouration made it possible to show help of electron microscopy, biochemical analysis, and also the otherwise invisible septa of Spirulina . Hindák ( 1985 ) recently, thanks to the support of molecular analysis, within assigned all planktonic forms of Arthrospira (including those the context of what is commonly referred to as a “polyphasic previously described as “S. platensis ”) in alkaline saline approach”. The genus Arthrospira has now become univer- lakes to “S. fusiformis” Voronichin 1934 (synonyms A. max- sally accepted, at least by taxonomists, as the name for the ima Setchell et Gardner 1917 , in Gardner 1917 , and A. gei- organisms used in mass culture. With the aim of further clari- tleri De Toni 1935 ) , since, in agreement with Fott and Karim fying the systematic position of the Arthrospira spp. com- ( 1973 ) , he considered “S . platensis ” to be a benthic species. monly used for mass culture operations (basically speaking, In order to distinguish Spirulina and Arthrospira in A. maxima and A. fusiformis), Komárek and Anagnostidis Bergey’s Manual of Systematic Bacteriology, Castenholz (2005 ) and Komárek and Hauer ( 2011) have recently urged a ( 1989) suggested the use of three features of helically coiled defi nitive abandoning of the commercial name Spirulina trichomes: (1) degree of inclination of the pitch of trichome ( Arthrospira ) platensis , recommending that it be replaced helix (from transverse axis); (2) aspect and visibility (LM) of with the taxonomically-correct name of A. fusiformis. cross-walls between the cells in the fi lament; (3) distribution The long story concerning the name Spirulina started (EM) of junctional pores in the cell wall. Thus Arthrospira about 130 years ago, when Wittrock and Nordstedt ( 1884 ) can be differentiated from Spirulina on the basis of the degree reported the occurrence in a stagnant pond near Montevideo of inclination of the pitch of the trichome helix, which forms of a blue-green alga with helical shaped fi laments, which an angle >45° from the transverse axis, the presence of easy they described as “Spirulina jenneri f. platensis ”, even visible septa and the distribution of junctional pores in one though it possessed septa. At that time the genus Spirulina circular row around the cross-walls. In 2001, with the Second 25 Arthrospira (Spirulina) 687

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)

Edition of Bergey’s Manual of Systematic Bacteriology, provides a summary of the synonyms of different Arthrospira Castenholz updated the key to separate Spirulina from species recognised by Komárek and Anagnostidis ( 2005 ) . Arthrospira. The two genera were distinguished according to However, this classi fi cation which is based only on phenotypic the trichome helix, cross-walls whether invisible or visible, aspects needs to be supported, particularly for the cultivated and cell diameter 2–4 m m in Spirulina versus typically strains, by molecular analysis. 6–12 m m in Arthrospira . In the past 20 years, the great development of molecular An important revision of the taxonomy and nomenclature biology allowed to accompany this modern techniques to the for “Spirulina platensis ”, “S. maxima ” and “S. fusiformis”, taxonomy based only on morphological features in accor- was suggested by Komárek and Lund (1990 ) who placed the dance with a combined methodology that Colwell (1970 ) for two taxa (“maxima ” and “fusiformis ”) within the planktonic the fi rst time indicated as the “polyphasic approach”. forms (i.e. with gas vacuoles). Furthermore, they suggested Viti et al. ( 1997) , using total DNA restriction profi le anal- that these two taxa, which may represent two species (A. fusi- ysis, carried out on 10 strains belonging to Arthrospira max- formis Komárek et Lund and A. maxima Setchell et Gardner), ima and Arthrospira platensis ( fusiformis), found a great have different geographical distributions. Hence, they consid- consensus between these the genotypic clusters and those ered A. maxima to be pantropical, while A. fusiformis was grouped using morphological criteria. Subsequently, limited to Africa and tropical and central Asia. Moreover, for Scheldeman et al. (1999 ) , by means of phylogenetic study the fi rst time they considered A. platensis (without gas vacu- using ARDRA (Ampli fi ed Ribosomal DNA Restriction oles, similar to A. jenneri ) to be a periphytic and benthic Analysis) of the ITS (Internal Transcribed Spacer) as a species that is essentially restricted to South America. molecular taxonomic marker, tested 51 Arthrospira cultures Subsequently, Desikachary and Jeeji Bai ( 1992 ) and Jeeji that theoretically represented 37 unique genotypes obtained Bai (1999 ) proposed that all the calyptrate forms of A. fusi- from different culture collections coming from four conti- formis should be included in the new species A. indica nents. The studies con fi rmed a clear separation of Spirulina Desikachary. This implied that the thickening of the apical from Arthrospira and the presence of two main genotype cell wall (calyptra) should be considered a signi fi cant taxo- clusters within Arthrospira which were unrelated as far as nomic feature (Fig. 25.12 ). Komárek and Anagnostidis the geographic origin of the strains, their denomination or (2005 ) concluded that Arthrospira has solitary and free- their phenotypic features. fl oating trichomes in the planktonic forms of subtropical and In 2002 Manen and Falquet investigated the genetic diver- tropical saline lakes (A. maxima and A. fusiformis ), while sity of 23 natural, cultivated and commercial strains of freshwater benthic forms were united in a fi ne, mostly slimy, Arthrospira by comparing the genus with 20 other non- thallus in A. platensis and A. jenneri. These authors placed Arthrospira cyanobacterial strains, utilizing the phycocyanin Arthrospira to the family Phormidiaceae (subfamily locus (cpcB-cpcA ). These studies confi rmed once again, that Phormioideae) and recognized 17 species worldwide accord- Arthrospira is not related to Spirulina and for the fi rst time ing based on phenotypic markers. The main morphological showed that the former genus constitutes a strongly sustained features of species without (i.e. benthic) gas vacuoles (ben- monophyletic group. Furthermore, the three genetically clus- thic) or with (i.e. planktonic) gas vacuoles, as distinguished tered lineages according to which Arthrospira was divided by Komárek and Anagnostidis ( 2005 ) and more recently by con fi rmed the general mismatch between their geographic Komárek and Hauer ( 2011 ) , ( http://www.cyanodb.cz). In the origin and morphology. Similar conclusions were reached by light of this new taxonomic grouping, the Table 25.2 Baurain et al. ( 2002) , who found no clear relationships 688 C. Sili et al.

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

between ITS clusters and the denomination, morphology and phycocyanin operon. It proved possible to divide the 53 origin of the strains. A phenotypic analysis on 35 (33 helical strains into two main clusters: cluster I comprising sequences and 2 straight) axenic and clonal Arthrospira strains was of American strains, and cluster II containing the sequences performed by Mühling et al. ( 2006 ) . The strains included of the strains originating from Asia and Africa. Both genetic 28 features, 14 of which were morphological, 1 ultrastructural, regions of the strains in this study showed a signi fi cant 7 physiological, and 6 biochemical. This study reported two sequence divergence among Arthrospira strains from East phenotypic clusters that had a high correlation to the charac- Africa, India and Mexico, indicating possible distinct evolu- ters describing the helical trichome shape: cluster I had a tionary lineages. It was concluded that there are deep geno- highly variable, always irregular, trichome helix that was typic divergences between Mexican and African/Indian dumbbell-shaped, or a fusiform trichome helix with a fast- strains of Arthrospira , which represent two distinct geno- diminishing helix attenuation toward the apices (A. maxima , types, which were also distinguishable on the basis of A. fusiformis , A. indica ), and cluster II with a regular trichome trichome morphology, and referable to the two tropical main helix and fast-diminishing helix attenuation toward the planktonic species A. fusiformis and A. maxima. apices ( A. platensis). These two clusters compared to the In conclusion, it appears clear that Arthrospir a is distinct molecular ones of the same strains reported by Scheldeman from Spirulina based on both morphological and molecular et al. (1999 ) and Baurain et al. (2002 ) showed a high rate criteria. However, because of the high morphological plastic- correlation. ity observed particularly with wild and clonal forms (i.e. Thirty-three new strains of Arthrospira isolated from laboratory and mass culture strains) of A. fusiformis, it is plankton sampled in Mexico, East Africa and India were sometimes diffi cult to distinguish the two edible species of investigated and compared with 53 strains or samples of ear- Arthrospira ( A. fusiformis and A. maxima ) by using only a lier studies (Dadheech et al. 2010 ) . The study included mor- morphological approach. The continuous advancements phological features and molecular phylogenetic analyses on achieved by molecular analysis, provided that the origin of the basis of nucleotide sequences of ITS between 16S rRNA strain is well known, can represent an important tool that and 23S rRNA genes and partial sequences of beta and alpha should contribute to better identifi cation of the Arthrospira subunits including in their genes spaces (cpc BA-IGS) of species. 25 Arthrospira (Spirulina) 689

1971) for tropical African lakes, and that by Busson (1971 ) , 25.4 Occurrence and Distribution who described the ecology of A. maxima as a food source in Mexico, together with its past and present use as a food Species of Arthrospira have been isolated mainly from alka- source. Other studies include those of Hindák (1985 ) , line, brackish and saline waters in tropical and semitropical Komárek and Lund (1990 ) and Jeeji Bai (1999 ) . regions (Castenholz 2001 ; Komárek and Anagnostidis 2005 ) . Most of the waterbodies in which A. fusiformis thrives are One of the striking facts about soda lakes is that, despite permanent or temporary saline alkaline lakes (natron lakes). apparently what might appear unfavourable conditions, these These are scattered in the hollows of the fossil dune system of environments are extremely productive because of high the Sahara and the Sudan-Sahel areas fed by aquifers and the ambient temperatures, high light irradiance and unlimited scanty rain (about 300 mm per year). The salinity of these −2 −1 supply of CO2 . Primary production rates up to 10 g m day lakes is highly variable: when evaporation exceeds in fl ow the have been recorded, which is about one order of magnitude salinity rises and leads to an accumulation of salt deposits on more than most productive streams and lakes in the world, the shores (Iltis 1971 ) . The mean water temperature is about making these the most productive aquatic environments any- 25°C, with oscillations of about ±10°C, although the temper- where. A characteristic feature of the majority of soda lakes ature in shallow lakes may reach 40°C during the warm sea- is the permanent or seasonal coloured blooms of phototrophic son. The mean daylight length is about 12 h and solar radiation microorganisms, as reported in a survey by Grant ( 2006 ) . reaches over 2,000 m mol photon m −2 s −1. The disposition and Though most records of Arthrospira species have been for slope of the impermeable clay and water-bearing sandy layers mass growths in alkaline water, some species have been favour subterranean seepage of water away from Lake Chad occur occasionally in freshwaters. Species with apparently (Maglione 1969). The relationships between the superfi cial more restricted distributions have been reported from pud- and the subterranean waters affect both the salinity and the dles (A. balkrishnaii ), fl owing and stagnant fresh water ionic composition. The latter is characterized by prevalence ( A. gigantea, A. jenneri ), springs ( A. massartii ), stagnant of monovalent ions, with Na+ much higher than K+ , and by water (A. desikacharyensis , A. gomontiana), stagnant and high concentrations of carbonate and bicarbonate, which lead sulphur-containing water (A. platensis ), ponds (A. khannae ), to pH values from 9.5 to 11.0. Ca2+ and Mg2+ concentrations 2− − fi lter beds ( A . tenuis), reservoirs ( A. santannae) and tanks are low. SO 4 concentration is usually high, while that of Cl ( A. joshi ). Less is known about these species, since they have is very low. The water of the lakes is often blue-green in not been used for mass cultivation. colour due to the mass development of phytoplankton. Among the various species, the most widely distributed, A. fusiformis is present as nearly monospecifi c popula- A. fusiformis (A. platensis sensu Rich 1931 ) , is mainly in Africa tions in the highly saline lakes (Iltis 1969a ) , only a few other (Chad, Kenya, Egypt, Ethiopia, Sudan, Libya, Algeria, Congo, phototrophs being found. In contrast, in the mesohaline Zaire, Zambia, Mozambique), Asia (Russia, Pakistan, India, lakes, Arthrospira co-exists with a number of other phototro- Sri Lanka, Myanmar, Thailand) and South America (Uruguay, phs, including other cyanobacteria, diatoms and Peru). A. maxima is in Central America (Mexico, California- dinofl agellates. Among the cyanobacteria, Anabaenopsis USA). Until a few years ago, this species was the main compo- arnoldii and many Oscillatoria spp. are often important. nent of the phytoplankton of the solar evaporation channels in Several Spirulina species may be present, especially S. laxis- Lake Texcoco (Mexico), while A. fusiformis dominated the sima , S. major and S. subsalsa. Studies by Margheri et al. alkaline saline lakes of the semi-desert Sudan-Sahel area ( 1975) of two African natron lakes, Lake Mombolo and Lake (Chad) and of the Rift Valleys (mainly Kenya). The frequent Rombou, in the region of Kanem north-east of Lake Chad, occurrence of A. fusiformis and A. maxima in saline, alkaline, found these Spirulina species as well as several Arthrospira tropical and subtropical waters has led to a widespread, but spp. Within the phototroph populations of both permanent possibly misleading, impression that these environments are and temporary lakes, the rotifer density can be very high, characteristic for the entire genus. The geographical distribu- although varying seasonally, reaching several hundred indi- tion of the main populations of Arthrospira is given in viduals per litre (Iltis and Riou-Duwat 1971 ) . In some lakes Table 25.3 . Apparently none have been reported from marine Arthrospira constitutes an excellent feed for the cichlid fi sh environments. However, when an adequate supply of bicarbon- Tilapia (Coe 1966 ; Bergman et al. 2003 ) . Another consumer ate, N and P, together with a suitable pH and salinity, are pro- of Arthrospira and other large fi lamentous cyanobacteria vided, Arthrospira species can be grown in seawater (Tredici such as Anabaenopsis is the fl amingo Phoenicopterus minor . et al. 1986 ) , although the biomass productivity attained was The pink colour of these birds comes from the carotenoid about 36% less than in Zarrouk’s medium. The biomass grown pigments originating in cyanobacteria (Fox 1976 ) . in seawater had a reduced protein content (13%). The detailed studies by Iltis established a direct correlation The most detailed studies on the ecology of Arthrospira between the biomass density of Arthrospira and the salinity are those for A. fusiformis by Iltis (1968, 1969a, b, 1970, of the water. The mass blooms occur at salinity values from 690 C. Sili et al.

Table 25.3 Geographical distribution of the main populations of Arthrospira Country Location Taxon References AFRICA Chad Natron lakes (Bodou, Mombolo, A. platensis, A. platensis f. minor Léonard and Compère ( 1967 ) , Rombou, Yoan) and pools (Latir, Iltis ( 1972 ) , and Rich (1931 ) Iseirom, Latir, Liva), Kanem region Lake Kailala, Lake Kossorom A. fusiformis Sili et al. (1999) Kenya Natron lakes A. platensis Rich ( 1931 ) and Vareschi (1982 ) (Bogoria, Crater, Elmenteita, Nakuru) Rift Valley A. fusiformis Ballot et al. ( 2004 ) Lake Bogoria A. platensis, A. platensis f . minor Tuite ( 1981 ) A. fusiformis Hindák ( 1985 ) Lake Simbi A. platensis Melack ( 1979 ) A. platensis = A. fusiformis Kebede and Ahlgren (1996 ) A. fusiformis Ballot et al. ( 2005 ) Lake Sonachi A. fusiformis Ballot et al. ( 2005 ) Lake Oloidien A. fusiformis Ballot et al. (2009 ) Ethiopia Lake Aranguadi A. platensis Melack (1979 ) Lake Chiltu, Green lake A. platensis = A. fusiformis Kebede and Ahlgren ( 1996 ) Egypt Lake Maryut A. platensis El-Bestawy et al. (1996 ) Sudan Lake Dariba, Jebel Marra A. geitleri Fott and Karim (1973 ) Algeria Pond Tamanrasset A. platensis Fox (1996 ) Congo Laka Mougounga Arthrospira sp. Fox ( 1996 ) Lake Kivu Arthrospira sp. Fox ( 1996 ) Zambia Lake Bangweolou Arthrospira sp. Fox ( 1996 ) Tunisia Lake Korba Arthrospira sp. Fathi et al. ( 2001 ) Mozanbique Wastewater ponds A. fusiformis Mussagy et al. (2006 ) South Africa Lake Tswaing A. fusiformis Oberholster et al. (2009 ) ASIA India Ponds A. maxima Desikachary and Jeeji Bai (1996 ) Lonar Lake; ponds; tank A. indica Desikachary and Jeeji Bai (1992 ) (Madurai, MCRC isolate) Myanmar Crater lake (Chapter 26) Arthrospira sp. Min Thein ( 1993 ) Pakistan Fish pond, Lahore Arthrospira sp. Fox (1996 ) Sri Lanka Lake Beria Arthrospira sp. Fox ( 1996 ) China Fish ponds, Nanking A. platensis Tsen and Chang (1990 ) Lake Bayannur A. platensis Zheng et al. ( 1992 ) Thailand Tapioca factory 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. platensisb Gomont ( 1892 ) EUROPE Spain Lake Santa Olalla Arthrospira sp. Rippka and Herdman (1992 ) France Tiny lake, Camargue Arthrospira sp. Fox (1996 ) Hungary Adasztevel-Oroshaz Arthrospira sp. Busson (1971 ) Romania Alkaline pond near Cluj-Napoca A. fusiformis Aldea et al. (2002 ) Serbia Salty puddles near river Tamiš A. fusiformis Fuzinato et al. (2010 ) a Reference strain PCC 7345 b Holotype 25 Arthrospira (Spirulina) 691

22 to 60 g L−1 , high carbonate-bicarbonate concentrations production facilities in the outside rim of the solar evapora- (pH 8.5–11.0) and temperatures from 25°C to 40°C. tor, where it was present in almost monospeci fi c populations, Generally, the waters populated by Arthrospira have a mean being associated with only a few other cyanobacteria such as salinity of 37 g L−1 . However, Arthrospira has been found at Synechocystis aquatilis. In 1994 Sosa Texoco ceased cultiva- salinity levels ranging from 8.5 to 200 g L −1 and in at least tion operations of Arthrospira maxima in the external ponds one case up to 270 g L−1 (Iltis 1968) . Biomass density (as dry of the “El Caracol” solar evaporator (Alcocer and Williams weight) can exceed 1 g L−1 . Sili et al. (1999 ) studied two soda 1996 ) . At present Lake Texococo is reduced to several sepa- lakes (L. Kailala, L. Kossorom) on the north-east fringe of rate waterbodies (Caracol, Lago recreativo and Nabor Lake Chad, where a number of small bays and inlets between Carillo) from where a number of A. maxima strains have the old sand dunes are located. Surveys showed that A. fusi- been isolated (Dadheech et al. 2010 ) . formis was clearly the dominant (Fig. 25.4 ), with some More recently, Arthrospira has been found occasionally fi laments of Anabaenopsis spp., Spirulina laxissima and in domestic wastewater treatment plants. in Maputu, Synecochoccus sp ., A. fusiformis is also found in some Monzambique (Mussagy et al. 2006 ) and Turkey (G. Torzillo, lakes of the northern Algerian Sahara having saline waters unpublished data), both characterised by pH ranging between (1–8%) and pH levels from 7 to 9. These waters, which have 7.1 and 7.7 and a conductivity between 1,000 and 2,000 m S −1 −1 −2 −1 lower alkalinity (about 20 meq L of HCO3 and CO3 ), but cm . In contrast to what has sometimes been believed, this Na+ levels similar to those of the highly alkaline saline suggests that Arthrospira can acclimate to new environments waters of the East African Rift Valley, may be considered to not markedly or exclusively alkaline. 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 25.5 Physiology effect on some of the Rift Valley lakes, where Arthrospira commonly is found, with a marked deterioration in their eco- The aim of this section is to point out relevant areas in which logical status and a progressive increase in nutrient concen- Arthrospira has been used as a model organism, together tration. The two lakes Naivasha an Oloiden (Kenia) are with other studies with data of signi fi cant importance in typical examples of this recent degradation and salinization understanding its growth, physiology and biochemistry, which has promoted the appearance of Arthrospira (Ballot especially when grown outdoors. One of the basic principles et al. 2009 ) . The Lake Oloidien hypereutrophic alkaline of microalgal technology is to re-create the environmental modi fi cation of the originally freshwater has caused a shift in and cultivation conditions permitting high productivity in a species dominance from coccoid Chlorophyceae to A. fusi- monoalgal culture. Without doubt light and temperature are formis and Anabaenopsis elenkinii . The main sources of the most important. In nature, the organism uses its gas vacu- nutrient input are cattle and goat herds, watering at the lake oles to regulate its position within the underwater light gradi- and local women washing clothes using detergents (Ballot ent and follow daily and seasonal changes in light fl ux. In et al. 2009 ) . shallow culture ponds, however, light availability has to be In Lake Nakuru, the dominance of A. fusiformis has been optimized by modifying operational parameters such as opti- documented since the 1960s (Talling and Talling 1965 ) . mal cell density and the degree of mixing and culture depth Okoth et al. (2009 ) demonstrated that A. fusiformis was the in order to obtain the highest productivity (Vonshak et al. most dominant species in terms of population density in 1982 ; Richmond 2004 b ; Belay 2008 ) . Lake Nakuru. However, the pollution effects due to the Adequate mixing coupled with maintenance of high cell urbanization process near the lake is expected to infl uence densities can to a certain extent reduce the effect of light satu- the lake’s phytoplankton community strongly in the future. ration of photosynthesis and damage to the photosynthetic As previously mentioned, a massive population of apparatus due to prolonged exposure to excess of light (pho- A. maxima was found in some of the external sectors of the toinhibition) (Torzillo and Vonshak 2003 ) . Suf fi cient mixing gigantic spiral-shaped solar evaporator used to extract salts also ensures an even distribution of nutrients and oxygen from the saline carbonate-bicarbonate rich waters of Lake degassing of the culture, and at the same time prevents Texcoco, Mexico. The evaporator lies 2,200 m above sea fi lament aggregation and sinking (Torzillo et al. 2003a ) .The level and is about 1 m deep. The alkaline (pH 10) saline fact that Arthrospira requires high alkalinity for growth water is moved slowly from the outer part of the evaporator ensures selective conditions in the growth medium and towards the centre (Gallegos 1993 ) . The water is high in Arthrospira grown in outdoor open ponds remains a quasi- − 2− − + HCO3 and CO3 , Cl and Na and the total salt concentra- monoculture (Vonshak et al. 1983) . Moreover, when other tion ranges from 11 to 39 g L −1 (Busson 1971) . In the past essential growth nutrients are present in suf ficient concen- the company that operated the plant, Sosa Texcoco (and trations, the elevated concentration of bicarbonate-carbonate later Spirulina Mexicana), had its “Spirulina” (A. maxima ) 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 diffi 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 −2 −1 the range of 150–200 m mol photon m s (Bocci et al. 1980 ); Fig. 25.13 Photosynthetic light response curves of A. fusiformis strain this value for irradiance is about one order of magnitude less M2 (○ photoinhibition; ● control) for 45 min at a PFD of 2,500 m mol than recorded outdoors in summer days (1,850–2,000 m mol photon m −2 s−1 photon m−2 s−1 ). By using a turbidostatic culture, a maximum −1 growth rate of 0.063 h was estimated for A. maxima strain Table 25.4 Photosynthesis-irradiance (P/I) parameters estimated from 4 Mx, isolated from Texoco Lake, Mexico, and 0.059 h −1 for Arthrospira fusiformis strain M2 cultures before and after 45 min pho- A. fusiformis strain K4, isolated Rombou Lake, Chad (Balloni toinhibition at a PFD of 2,500 m mol photon m−2 s−1 at 35°C et al. 1980 ) , which correspond to doubling times of 11 and Parameter Unit Control Photoinhibited −1 −1 11.7 h, and a light transformation effi ciency of 7.2% and Pmax m mol O 2 mg chl h 800 600 −2 −1 7.8% of the photosynthetically active radiation (PAR, 400– I K m mol photon m s 430 610

700 nm) for A. maxima and A. fusiformis, respectively (Bocci a m mol O 2 (mg chl m mol 1.915 1.094 −1 photon m −2 s−1 )−1 h −1 et al. 1980 ) . Based on a requirement of 20 quanta mol CO 2 , R m mol O mg chl −1 h −1 −20.5 −26.5 Ogawa and Aiba ( 1978 ) estimated the slightly higher value 2 for photosynthetic ef fi ciency of 11.4% PAR. P max light saturated rate, I k saturation irradiance, a initial slope of the P/I curve, R dark respiration rate 25.5.1.2 Light Stress – Photoinhibition Different strains of Arthrospira may differ in their sensitivity a stronger effect on the initial slope of the P/I curve i.e. on to light stress (Vonshak et al. 1988b ; Vonshak and Novoplansky light-limited photosynthetic activity. In contrast to higher 2008 ) . Photoinhibition, which is the loss of photosynthetic plants and microalgae, in the case of cyanobacteria it has capacity due to damage to photosystem II (PSII) caused by always assumed do not use an antenna-related quenching photon fl ux densities (PFD) in excess of those required to mechanism to decrease the amount of energy funneled to saturate photosynthesis, can be manifested in various ways. photosystem II (PSII) reaction centre (Campbell et al. 1998 ). These include a decrease in the maximum quantum yield of Recently, however, evidence has been presented for the exis-

CO2 uptake or O2 evolution, a decrease in the convexity of the tence of at least three distinct mechanisms for dissipating photosynthesis light response curve (P/I), and, in the case of excess energy in cyanobacteria. One of these photoprotective prolonged exposure to excessive light, a decrease in the light- mechanism is related to the phycobilisomes, the extramem- saturated photosynthesis (Leverenz et al. 1990 ; Long et al. branal antenna of cyanobacteria PSII. In this photoprotective 1994 ) . In at least one A. fusiformis, strain P4P, photoinhibi- mechanism the orange carotenoid protein (OCP) in tion was probably due to a change in the turnover rate of a Synechocystis sp PCC 6803 plays an important role specifi c protein, D1, which is part of PSII. Observed differ- (Kirilowsky 2007 ; Wilson et al. 2006). The Arthrospira max- ences may be genotypic or environmental responses. Cultures ima OCP contains 3’-hydroxyechinenone (Kerfeld 2004 ). acclimated to high light intensities exhibit a higher resistance However, mechanism of this novel photoprotective process in to photoinhibition (Vonshak et al. 1996a ) . In a study of the cyanobacteria awaits elucidation. photosynthetic light response curve of A. fusiformis strain M2 cells exposed to strong light (Torzillo and Vonshak 1994 ) , 25.5.1.3 Effect of Temperature on Photosynthesis photoinhibition caused a 48% reduction in the initial slope of and Respiration the P/I curve compared to the control (Fig. 25.13 ). However, A detailed study of the response of an A. fusiformis strain there was less effect on Pmax (maximum saturation rate), which M2, to temperature was performed by Torzillo and Vonshak was reduced by about 25% (Table 25.4 ). These results empha- ( 1994) . They determined the optimal temperature for photo- size that photoinhibition not only affects Pmax, but actually has synthesis, and also measured the effect of temperature on the 25 Arthrospira (Spirulina) 693

Fig. 25.15 Temperature responses of three Arthrospira fusiformis iso- lates measured as the increase in chlorophyll concentration after incu- bating the cells for 4 days on a temperature block under constant light (100 m mol photon m −2 s−1 )

during the daylight period may be lost through respiration at night (Guterman et al. 1989 ) . Torzillo et al. (1991 ) concluded Fig. 25.14 Effect of temperature on photosynthesis (a ), and dark that night biomass loss due to dark respiration depended on respiration (b ) rates of A. fusiformis strain M2. Cells were allowed to the temperature and light irradiance to which the cultures equilibrate for 15 min at each temperature before measurement 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 dark respiration rate by following the O2 uptake rate in the revealed that the protein content was ca. 57% and 71%, of dark. The optimal temperature for photosynthesis was 35°C dry weight respectively, whereas the carbohydrate content (Fig. 25.14a ), while dark respiration was highest at 45°C during the same periods was ca. 34% and 19%, of dry weight (Fig. 25.14b ). At temperature of 50°C the respiration rate respectively. An increase in carbohydrate in cells was observed dropped to almost zero. At temperature extremes outside when these were grown at the suboptimal temperature of those optimal for growth, both respiratory and photosynthetic 25°C. Excess carbohydrate synthesized during the day was activity declined. However, the sensitivity of respiration to then partially used for protein synthesis at night, while most such extremes was signi fi cantly greater than the sensitivity of them were lost by respiration (Torzillo et al. 1991 ) . of photosynthesis under the same conditions. Under conditions where respiration was completely inhibited, photosynthetic 25.5.1.4 Effect of Temperature on Growth oxygen evolution was maintained at about 30% of the optimum and Cell Composition value. A temperature-dependent exponential relationship In nature Arthrospira is found in permanent or temporary between 15°C and 45°C was obtained, with the respiration water bodies at relatively high temperatures. The optimal rate increasing as temperature increased. The temperature- temperature for laboratory cultivation of this organism is dependent dark respiration rate is given by: about 35°C. However, many Arthrospira strains differ in their optimal growth temperature as well as in their sensitiv- R= 0.771.e (0.0636.T) ity to extreme values. For instance, in the comparison of strains shown in Fig. 25.15 , strain SA2 has a relatively low −1 −1 where R is the respiration rate (m mol O2 mg chl h ) and T optimal growth temperature (24–28°C), while the strain EY is the temperature (°C). An Arrhenius plot for respiration grew well up to 38–40°C. The isolate marked K was charac- showed an activation energy of 48.8 kJ mol−1 for Arthrospira . terized by a relatively wide range of optimal growth tem-

The temperature coef fi cient (Q10 ) calculated for the 15–45°C peratures: 28–36°C. This is just one example of the variations range was 1.85. The respiration-to-photosynthesis ratio in that exist among the different strains of Arthrospira , as Arthrospira was 1% at 20°C and 4.6% at 45°C (Torzillo and regards optimal growth temperatures. Signi fi cant differences Vonshak 1994 ) . These low values confi rmed the general in the maximum temperature for growth have been also assumption that cyanobacteria have low respiration rates found within the two species of A. maxima and A. fusiformis . (van Liere and Mur 1979 ) . However, the respiration-to- A. maxima strains were apparently unable to grow above photosynthesis ratios measured in these experiments were 36°C, while some A. fusiformis strains were found to be found to be much lower than those reported for outdoor cul- capable of growing at a temperatures higher than 40°C tures of Arthrospira , where up to 34% of the biomass produced (Tomaselli et al. 1987 ) . Arthrospira fusiformis strain M2 was 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 ). Fig. 25.16 Effect of oxygen concentration and temperature on maximum quantum yield of PSII (Fv /Fm ) of Arthrospira fusiformis strain M2 cultures grown outdoors in photobioreactors. (■) Low oxygen- optimal 25.5.1.5 Interaction of Low Temperature with Light temperature; (▲) high oxygen – optimal temperature; (●) high oxygen-low and High Oxygen Concentration temperature; (Δ) PFD 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 sity is relatively low, as demonstrated by several experiments handle excessive light energy, since they have a higher rate carried out both in the laboratory and outdoors (Torzillo et al. of electron transport, an active repair mechanism and more 1984 ; Marquez et al. 1995 ; Vonshak et al. 1996a ) . The com- ef fi cient means of energy dissipation (Vonshak 1997b ) . The bination of high oxygen concentration and high light inten- saturation pulse method was applied in order to study the sity can be a very common event in outdoor cultures, synergistic effect of high oxygen concentration and low tem- particularly in closed systems. For example, in well growing perature in outdoor cultures of A. fusiformis strain M2 A. fusiformis strain M2 cultures in tubular photobioreactors (Torzillo et al. 1998 ) . It was observed that the combination of (i.d. 5 cm), the oxygen concentration can increase at a rate of low temperature (25°C) and high oxygen concentration (70– 2–3 mg L −1 min −1. This results in an oxygen concentration of 80 mg L−1 ) had considerable impact of PSII photoinhibition up to 70–80 mg L−1 (Torzillo et al. 1998 ) . evaluated as changes in the Fv /Fm ratio (variable to maximum Sensitivity to stress is strongly in fl uenced by the light

fl uorescence). The Fv /Fm ratio decreased down to 64% of the acclimation conditions of the cells. Cells acclimated at low morning value in the middle of day in the high oxygen culture light usually saturate photosynthesis at lower light intensities (Fig. 25.16 ). When high oxygen concentration was combined (Ramus 1981 ) . Measurements of chlorophyll fl uorescence with low temperature, the Fv /Fm ratio decreased to up to 45% quenching in outdoor cultures of Arthrospira showed that of the morning value. Complete recovery of the F v /F m ratio sensitivity to photoinhibition strongly increased in A. fusi- occurred in the afternoon only in the low oxygen cultures, while formis strain M2 cultures acclimated to low light. Yet, despite it reached about 80% of the morning value in the high oxy- a 40% reduction in the Fv /Fm ratio observed in cells exposed gen culture, and only 63% in the culture exposed to combina- to high stress conditions i.e. a combination of suboptimal tion of high oxygen and low temperature (Fig. 25.15 ). temperature (25°C) and high dissolved oxygen concentra- Photoinhibition studies in the cyanobacterium Synechocystis tions (up to 60 mg L−1 ), no signifi cant loss in the D1 content sp. PCC 6803 have indicated that photodamage is initiated was found. By contrast, under the same stress conditions, the by the direct effect of light on the oxygen evolving complex D1 content was reduced by 50% in low-light acclimated cul- (OEC) and that reactive oxygen species (ROS) inhibit the tures (Torzillo et al. 2003a ) . Productivity of low-light accli- repair of photodamaged photosystem II primarily suppress- mated cultures was lower than that obtained in high-light ing the synthesis of proteins de novo (Murata et al. 2007 ). acclimated cultures. Light acclimation also strongly The photosynthetic electron transport system represents infl uenced the effect of stress on the pigment composition of the major source of ROS, that is, singlet oxygen, hydrogen the A. fusiformis strain M2 cells. Pigment analysis showed peroxide and the superoxide radical (Asada 1994 ; Krause that ß-carotene decreased during the day in high stress cul- 1994 ) . When scavenging of potentially damaging oxygen spe- tures, while the mixoxanthophyll and zeaxanthin content cies is insuffi cient, photoinhibition can occur. High oxygen increased. The considerable presence of mixoxanthophyll concentration itself can infl uence the growth and the photo- and zeaxanthin may provide a source of defense for cells synthetic activity of the cultures even when the light inten- against photooxidation (Torzillo et al. 2003a ) . 25 Arthrospira (Spirulina) 695

25.5.2 Effect of Salinity on Growth, High rates of dark respiration in cyanobacteria due to Photosynthesis and Respiration salinity stress had been reported previously (Vonshak and Richmond 1981 ; Fry et al. 1986 ; Molitor et al. 1986 ) . This The occurrence of Arthrospira spp. in marine habitats has high activity may be associated with the increased level of not been documented, at least not as dominant blooms like maintenance energy required for pumping out the excess of those found in the natron lakes. This is most likely due more sodium ions. Variable chlorophyll fl uorescence was applied to the very low content of bicarbonate rather than to the high by Lu et al. (1999 ) in order to study the kinetics of the response content of NaCl. Arthrospira , unlike marine organisms, does of A. fusiformis strain M2 cells to salinity stress. The study not require high NaCl concentration for growth; rather, it revealed that the response of PSII photochemistry during the only tolerates the salt. The exposure of Arthrospira cultures fi rst 12 h of their exposure to high salinity consisted of two to high NaCl concentrations at fi rst results in an immediate phases. The fi rst phase, which took place during the fi rst 4 h, cessation of growth; after a lag period, a new steady state was characterized by an immediate drop in Fv /F m during the of growth is established (Vonshak et al. 1988a ) . The length fi rst 15 min (about 26%) after exposure. This was followed by of the time lag is exponentially correlated to the degree of a recovery to around 92% of the original level after 2 h incu- salinity stress imposed on the cells. In many cases, this lag bation. After 4 h from the beginning of the salt treatment, is associated with a decline in chlorophyll and biomass another decline in Fv /F m was observed, which reached about concentrations in the culture (Vonshak et al. 1988a ) . 70% of the initial value after 12 h (Lu et al. 1999 ) . According When the biomass composition of two strains grown to Lu and Vonshak ( 2002 ) , salt stress in A. fusiformis strain under different salt stress conditions was compared, M2 inhibited the electron transport at both the donor and signifi cant changes, mainly an increase in carbohydrate and acceptor sides of PSII, and resulted in damage to the phyco- a decrease in the protein levels, were observed. These bilisomes and a partial disconnection of these from PSII changes correlated with the degree of stress imposed (i.e. which shifted the distribution of excitation energy in favour higher level of carbohydrate at a higher salt concentration). of the PSI (state 2). An alteration in the structure of the phy- The difference in the level of carbohydrate accumulated by cobilisomes of A. fusiformis in response to an enhanced Na + the two strains may also refl ect a difference in their ability to level was also reported by Verma and Mohanty ( 2000) . The acclimate to salt stress. Changes in lipid synthesis have also effect of salt stress on A. fusiformis was further investigated been demonstrated: in salt-stressed cells, with an increase in by Gong et al. (2008 ) . The results confi rmed previous obser- lipid content and in the degree of fatty acid saturation is vations that salt stress leads to damage on the reducing side of observed with speci fi c modi fi cations in the long-chain fatty PSII and, in particular, to a modi fi cation of the QB niche. acids (C18). In A. fusiformis strain M2 the oleic acid content Moreover, the authors con fi rmed that also the donor side of doubled when the organism was grown in presence of 0.5 M PSII was affected by salt stress, and that this damage was NaCl (Tomaselli et al. 1993 ) . associated with a dissociation of the PsbO protein from the It has been suggested that exposure to high salinity is thylakoid membranes (Gong et al. 2008 ) . accompanied by a higher demand for maintenance energy by Another modi fi cation in salt-stressed cells is an increase in the stressed cells (Blumwald and Tel-Or 1982 ) . Changes in the respiration rate (Vonshak et al. 1988a ) . In A. fusiformis the photosynthetic and respiratory activities of an Arthrospira strain M2, this increase was two- and three-fold when the con- strain were measured over a period of 48 h beginning 30 min centration of salt in the medium was increased from 0.5 to after exposure to 0.5 and 1.0 M NaCl. A marked decrease in 0.75 mM, respectively (Zeng and Vonshak 1998 ) . Sensitivity to the photosynthetic oxygen evolution rate was observed photoinhibition was found to be enhanced when A. fusiformis 30 min after exposure to the salt. This decline was followed strain M2 cells were exposed to sizable combinations of high by a recovery period, characterized by a lower steady state light and salt stress (Zeng and Vonshak 1998 ) . It seems that rate of photosynthesis (Vonshak et al. 1988a ) . The immedi- many cyanobacteria are capable of compensating the reduction ate inhibition of the photosynthetic and respiratory systems in the energy supply coming from the photosynthetic pathway after exposure to salt stress has been explained by Ehrenfeld by signi fi cantly increasing their respiratory activity, which in and Cousin ( 1984 ) and Reed et al. ( 1985 ) , in, respectively, addition can provide carbon skeletons for the synthesis of Dunaliella and in Synechocystis. A short-term increase in organic osmolytes and for the extrusion of Na+ in cells in order cellular sodium concentration was due to a transient increase to maintain the osmotic balance (Peschek et al. 1994 ) . 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 25.5.3 Osmoregulation rapid entry of sodium, might be the result of the detachment of phycobilisomes from the thylakoid membranes (Blumwald During the course of acclimation to salinity, an osmotic et al. 1984 ) . adjustment is required (Borowitzka 1986 ). In Arthrospira 696 C. Sili et al. this involves the accumulation of low molecular weight car- values of 10.0 and 11.5, the intracellular pH values were only bohydrates. These have been identifi ed as a nine carbon het- 8.0 and 8.5, respectively. eroside called glucosyl-glycerol, plus a trehalose (Martel Many alkaliphiles require sodium in order to survive in et al. 1992 ) . Changes in the amount of cellular trehalose and extreme alkaline environments (Horikoshi and Akiba 1982 ) . in the activity of the enzyme malthooligosyl trehalose hydro- Padan et al. ( 1981 ) demonstrated that an active sodium – pro- lase (Mth), the enzyme that catalyses the formation of treha- ton antiporter is required in order to maintain a low internal lose from maltooligosyl trehalose, were recently investigated pH. Most cyanobacteria require sodium in an mM or sub- in A. fusiformis by Ohmori et al. ( 2009 ) . These authors millimolar concentration range in order to maintain viability, observed that the amount of trehalose in the cells increased especially at an alkaline pH (Miller et al. 1984 ; Espie et al. rapidly when a high concentration of NaCl was added to the 1988) . Some reports have indicated, in cyanobacteria incubated culture medium. The inhibition of sodium ion transport by in a sodium-defi cient media, the loss of the photosynthetic means of amiloride, a sodium channel blocker, and mon- oxygen evolution (Zhao and Briand 1988 ) and a degradation ensin, a cation ionophore, signi fi cantly decreased the amount of the thylakoid membranes (Averdano et al. 1989 ) . The effect of cellular trehalose, suggesting that the in fl ux of Na+ into of sodium deprivation is very dramatic in A. fusiformis . the cells was connected with an accumulation of trehalose. In this obligate alkaliphilic cyanobacterium, a strict Na The synthesis of trehalose was comparable when KCl was dependence has been found for both the growth and photo- used to replace NaCl, while the effect on cellular trehalose synthetic activity of the cells (Schlesinger et al. 1996 ) . It has synthesis was only half as much by the same amount of LiCl. been demonstrated that concentrations of Na+ no less than It was suggested that the cells accumulated trehalose mainly 50 mM in the medium are required in order to maintain via ionic stress, rather than by osmotic stress. It was also sug- growth at a high pH. Depletion of Na+ causes a light-depen- gested that the infl ux of Na+ , and not the mere presence of dent inhibition of PSII and a loss in the phycocyanin content Na+ in the medium, is connected with the physiological effect of A. fusiformis cells (Schlesinger et al. 1996 ) . It has been of NaCl. It was concluded that the addition of NaCl activated suggested that pigment bleaching and a loss of PSII activity Mth and, at the same time, triggered a transcription of the are due to the inability on the part of the cells to maintain an mth gene. Disruption of the mth gene, if such is possible, appropriate intracellular pH. Pogoryelov et al. (2003 ) theo- may provide further evidence on the role of Mth in the stress- rized that the specifi c site of inactivation of the photosyn- response accumulation of trehalose in Arthrospira . 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 25.5.4 Arthrospira as an Alkaliphile the dark and for short periods, without any detectable dam- age to the photosynthetic chain. A very intriguing question is Most Arthrospira species currently grown in mass culture how Arthrospira is capable of coping simultaneously with were isolated from alkaline and saline or brackish waters two extreme environmental situations: a high pH and a high characterized by high levels of carbonate-bicarbonate and concentration of Na+ . In fact, not only does the adaptation of high pH levels (Sect. 25.4 ). The physiological characteristics cells in this organism permit growth at high pH and high Na+ which enable a microorganism to thrive in such an extreme concentrations, as in the case of many other alkali-tolerant environment are still not fully understood. Arthrospira not cyanobacteria, but also the cell functions are optimized to only survives at high pH values, but actually thrives in such these extreme conditions (pH 9.5–11.5 and 150–250 mM conditions. Belkin and Boussiba (1991 ) , who compared NaCl). Further studies on the energy requirement and the growth pH optima for the two cyanobacteria Anabaena involvement of light in the regulatory process are needed. 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 25.5.5 How Does Arthrospira Compete range. When incubated at pH 7.0, the growth rate of in Culture? Arthrospira was severely inhibited and was only 20% of that under the optimal conditions. This high pH (>8) requirement Studies performed on different Arthrospira isolates from clearly de fi nes Arthrospira as an obligatory alkaliphile various sources demonstrate highly variable responses to dif- (Grant et al. 1990 ) . ferent ecological factors (Tomaselli et al. 1987 ; Vonshak One of the major problems faced by cells in a highly alka- et al. 1996a, b ) . It is on the basis of this variability that strains line environment is that of regulating their internal pH. suitable for the biotechnological applications are selected: Belkin and Boussiba (1991 ) were the fi rst to measure the i.e. photo-, thermo- or osmotolerant strains (Tomaselli et al. ability of Arthrospira to maintain a pH gradient across its 1993; Vonshak and Guy 1992 ; Vonshak et al. 1988b ) . cytoplasmic membrane. It was shown that, at external pH Detailed studies aimed at measuring the ecological speci fi city 25 Arthrospira (Spirulina) 697 of the different species, particularly of those utilized for total potential of the different sites in China now exceeds commercial purposes, are scarce (Kebede and Ahlgren 1996 ) . 2,000 t (dw), but the situation is changing rapidly due to the Nevertheless, full knowledge of the ecological demands of development of production on the Ordos Plateau of Inner the chosen species and the use of more productive selected Mongolia (Lu et al. 2011 ) . Although the average annual tem- strains is important if high productivity is to be achieved in perature in the region is only 6.4°C (Qiao et al. 2001 ) , which intensive outdoor cultivation systems. The use by commer- poses a challenge (Li et al. 2003 ) , commercial production cial producers of multistrain inocula which respond posi- under greenhouses permits lower costs than elsewhere in tively to changes in abiotic factors (due to their heterogeneity) China and it is aimed to reach an annual output of 3,000 t represents another way of compensating for the present lack within a few years. Lu et al. ( 2011 ) state that a 1,000,000-t of well-characterized strains. plan for production has been sketched out. Like other components of the phytoplankton, Arthrospira Arthrospira production is mostly carried out in raceway plays a role in cycling nutritional elements and in their ponds of 2,000–5,000 m2 and may contain between 400 and passage into different food chains; moreover, it provides an 1,000 m 3 of culture according to the depth adopted, which important direct food source for zooplankton, fi shes, birds can vary between 15 and 40 cm depending on season, desired and humans. The same factors that in the original habitat algal density and, to a certain extent, the desired biochemical regulate the occurrence of Arthrospira in monospeci fi c pop- composition of the fi nal product (Shimamatsu 2004 ; Belay ulations are those required for both successfully maintaining 2008 ) . Further information on commercial production of a monoculture in intensive production ponds outdoors and Arthrospira (Spirulina) the reader can be found in more spe- for achieving high productivity. Factors such as high alkalin- cialized books (Vonshak 1997b ; Richmond 2004a ; Gershwin ity and salinity are the key features that selectively limit the and Belay 2008 ) . growth of other organisms and predators. Moreover, once the The major share of the market for this organism is for optimal conditions for the growth of Arthrospira are main- health food involving crude biomass production. This has the tained in ponds, this organism successfully outcompetes advantages of simple processing (harvest and rudimentary other occasionally occurring phototrophs for nutrient and handling) keeping production costs reasonably low, and to light. This may be attributable to its fast growth rate and pho- elude the competition with chemical industry which cannot toacclimation capability, and its high tolerance to environ- match the wealth in nutritional bioactive components and mental stress (irradiance, salinity, temperature). attractiveness of natural products (Boussiba and Affalo 2005 ) . The experience gathered over many years of industrial production of Arthrospira has allowed to individuate the 25.6 Mass Cultivation of Arthrospira main problems related with commercial scale production (Shimamatsu 2004 ) . They are: (1) how to maintain an unial- At present Arthrospira (commercially indicated as Spirulina) gal production, that is, without sizeable presence of contami- represents the second most important commercial microalga nants all over the year, and (2) how to maintain a consistent in term of US$ (after Chlorella ), while in terms of total bio- quality of the product. These two aspects are strongly depen- mass produced, the Arthrospira market is twice or more of dent on an appropriate strain selection and adequate formula- that occupied by Chlorella (Torzillo and Vonshak 2003 ) . tion of the culture medium. Major criteria for the selection of Commercial production takes two broad approaches: that of strains are growth rate, biochemical composition and resis- industrialised countries who interested in producing the bio- tance to environmental stress, usually high light and low and mass for health food market, as well as for the extraction of high temperatures at each production site. The chemical fi ne chemicals, and that of developing countries looking for composition of culture medium is similar to than used for a rich source of protein which can be produced locally, using laboratory culture (Zarrouk’s medium) with a high content −1 −1 marginal land and saline water unsuitable agriculture, and of NaHCO 3 (16.8 g L ), K2 HPO4 (0.5 g L ) and KNO 3 −1 the opportunity for treating animal and human waste (2.5 g L ), pH is maintained at 9.5–10.3 by arti fi cial CO2 (Laliberté et al. 1997 ; Habib et al. 2008 ) . A major producer supply. The high alkalinity and high pH represent an ef fi cient of Arthrospira is the DIC group of companies, Earthrise in barrier against contamination by other microalgae, permit- California, USA, Hainan DIC Marketing in Hainan Island, ting the maintenance of a unialgal culture in open ponds. The China. These facilities produce about 1,000 t Arthrospira cost of nutrients usually dictates the choice of nutrient to be annually (Belay 2008 ) . Another important Arthrospira pro- used. For example, the use of ammonia, which is cheaper ducer is Cyanotech Corporation of Hawaii with an annual than nitrate, is restricted by the fact that it can be toxic to the + production of 300 t (dw). Other producers are located mainly cultures. The predominance of NH3 over NH4 at pH values in the Asia-Pacifi c region, particularly China and India (Lee greater than 9.25 (the pKa of the ammonia system at 25°C),

1997 ) and the highest production capacity of Arthrospira together to the high permeability of the NH 3, causes uncou- biomass takes place in China. Recent estimates are that the 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 measurements such as chlorophyll fl uorescence technique considered optimal for the growth of Arthrospira, concentra- (Sect. 25.5.1 ), and to reduce as much as possible the stress tion of free ammonia above 2.5 mM is reported to be toxic to damage mechanically created by excessive stirring or by use many algae (Abeliovich and Azov 1976 ; G. Torzillo, unpub- of centrifuge pumps for culture transfer (e.g. during the har- lished data). For this reason, the fed-batch addition of vesting). In most cases, the steps which proved effective in ammonia-based nitrogen sources is mandatory to effectively preventing contamination by Chlorella were: maintaining a prevent any inhibiting effect (Rodrigues et al. 2011 ). high bicarbonate concentration (e.g. 0.2 M); and taking pre- Moreover, the fed-back addition of ammonia-nitrogen can cautions to maintain the dissolved organic load in the culture also reduce the risk of nitrogen defi ciency which may occur medium as low as possible. Amoeba grazing on Chlorella, when ammonium salts are used as the only source of nitro- and Arthrospira have been also observed in some improperly gen and ammonium is lost by out-gassing. For this reason the operated commercial ponds. Addition of ammonia (2 mM) simultaneous use of nitrates and ammonium salts are often arrested the development of these grazers (Vonshak et al. used Arthrospira farms to avoid the risk of nitrogen 1983 ) . Similar observations have been reported by Lincoln depletion. et al. (1983 ) , who showed that the population of grazers was Another important nutrient component of the culture signifi cantly reduced when ammonia was used as the main N medium is the carbon source. The carbon content of the source. However, ecological control by predators, although Arthrospira biomass is about 50% (Torzillo et al. 1991 ), this has occasionally been observed in natural lakes domi- meaning that a minimum of 1.8 g of CO2 to synthesize 1 g nated by Arthrospira (e.g. Lake Ankorongo, Madagascar), (dry weight) of biomass is required. Therefore, the utiliza- has been never applied in industrial cultures (Shimamatsu tion of CO2 in the fl ue gas may help to reduce the cost of the 2004 ) . biomass production (Ferreira et al. 2012 ). Strategies for sup- Safety and quality control of biomass represent another plying CO2 in large-scale cultivation ponds to increase the important issue for the industrial production of Arthrospira

CO2 absorptivity have been proposed (Bao et al. 2012 ). (Belay 2008 ) . Toxic species of other cyanobacteria may be Cultures are operated according to a semi-continuous present (Chap. 24 ) and some Arthrospira biomass companies regime where a constant cell concentration is maintained in have developed methods for their determination and actually the ponds, usually in the range of 400–600 mg L−1 dry weight, certify their products to be free of toxins, such as microcys- by daily harvesting of biomass to the extent that it has grown tins. Arthrospira biomass does not contain microcystin (An over the last 24 h. This concentration range is mainly dictated and Carmichael 1996 ) , but contamination by other cyanobac- by harvesting effi ciency. However, a concentration below teria cannot be ruled out. A potential problem of open ponds 100 mg L −1 dry weight can make cells susceptible to photo- of Arthrospira production is that the water may be contami- inhibition (Vonshak and Guy 1992 ; Torzillo et al. 1998 ) . nated with pathogenic organisms. Handling of the product This phenomenon can occur at the start of the pond inocu- during processing can also result in microbial contamination lation, when the culture is too dilute and not suf fi ciently (Belay 2008 ) . Production cost is an area where little infor- acclimated to high light conditions, or when cultures are mation is available as companies are usually not available to subject to a combination of high light and low temperature provide this information. However, cost of production is esti- (Sect. 25.5.1 ). mated to range from US$ 6–12 kg −1 dry matter. The lower Maintaining a culture in large open ponds without any fi gure is based on a “feed” grade product, not for direct contamination by other organisms is one of the major chal- human consumption. The product is sun-dried and contains a lenge. Vonshak and Richmond ( 1988 ) estimated the overall high ash and low chlorophyll content. The demand for such annual loss of productivity due to contamination and subse- a product is continually growing, as its relatively low price is quent discarding of culture to be in the order of 15–20%. making it more attractive to the feed industry as an additive Dilution of the cultures caused by rainfall and low light con- to fi sh and chicken feed. This demand is refl ected in the ditions often facilitates the onset of microalgae (Chlorella in reports of an increase in the production of feed grade prod- particular), protozoa and insects. Continuous recycling of ucts up to 60 t year −1 by Earthrise Farms, compared to only a medium can easily result both in the concentration of con- few tonnes of feed grade produced some years ago. A starch- taminants like Chlorella or Arthrospira strains characterized producing factory in Thailand has started to use its water by having short fi laments, that are not arrested by the fi lters, refuse from an anaerobic digester to grow Arthrospira bio- and in the accumulation of organic matter because of decom- mass and then sun-dry the product. Cost estimates indicate position of death algae. This situation usually stimulates the that the product can be sold for less than US$ 6 kg −1. This is growth of graze particularly protozoa. In order to prevent this a very important step towards the production of Arthrospira situation it is mandatory to maintain the cultures at the opti- biomass, not only as a health food product, but for a much mum growth conditions, by continuous monitoring of the larger market such as for feed additives. Further reduction of physiological state of the cultures using fast and reliable production cost depends upon several factors: (1) increase in 25 Arthrospira (Spirulina) 699

Fig. 25.17 (a ) An overview of the soda Lake Kossorom (Chad); (b ) a formis blooms from the surface of Lake Kossorom; (e ) young women bloom of Arthrospira fusiformis (Lake Kossorom); (c ) accumulation of collecting pieces of sun-dried Arthrospira biomass (dihé ) after drying A. fusiformis biomass at the edge of the lake; ( d) harvesting A. fusi- them on sand (Photos M.R. Tredici) productivity of the organism; (2) control of contamination mass into a variety of food products such as granola bars and and hence the reduction on the culture renewable time; (3) various kinds of pasta. In Mexico and China, subsidized by increasing of harvesting effi ciency; (4) reduction of the over- the government, Arthrospira powder is added to child food all operational ef fi ciency of the farm (Shimamatsu 2004 ) . 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 25.7 Market and Application under the “Lima Blue” brand name (Dainippon Ink 1980, 1981 ) . The product is mainly used as a colourant for the food The biomass produced is mainly sold to the health food addi- market to provide an edible dye for ice cream and as a natural tives market in the form of powder or pills. It has also been dye in the cosmetics industry. The main problem is that the selected by the NASA (National Aeronautics and Space pigment is light sensitive and special care has to be taken in Administration) and by the European Space Agency as one handling the dye to protect it from bleaching. Full accounts of the primary foods for long-term space missions. In many of the potential application of Arthrospira as a nutritional cases the term “Spirulina” has become a practical synonym and therapeutic supplement in health management are given for cultivated microalgae. Attempts have been made by by Belay (2002 ) and Gershwin and Belay (2008 ) . Proteous (a marketing company mainly associated with Beside its commercial production, Arthrospira still has an Earthrise Farms in the USA) to incorporate Arthrospira bio- important role as food in the economy of some African 700 C. Sili et al.

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)

countries. In 2000 a delegation funded by the African dihé annually harvested from Lake Kossorom (about 40 t) Development Bank visited a number of African countries in amounted to more than US $ 100,000 which represents an an attempt to study to what extent the local tribes use important income for the economy of the area (Fig. 25.17 ). Arthrospira biomass as a part of their local food in order to Natural production of Arthrospira has been also documented supplement their protein requirements (Sodelac 2000 ) . The in Myanmar. Four volcanic lakes with natural Arthrospira internal report issued indicated that the harvesting of natu- bloom were studied beginning in 1984. Production began at rally-occurring blooms of Arthrospira from lakes around the Twin Taung Lake in 1988, and in 1999 increased to Rift Valley is a very common tradition. A well-de fi ned social 100 t year−1 . About 60% is harvested from boats on the sur- and practical structure has been reported. Only Kanembu face of the lake, while the other 40% is cultivated in outdoor women carry out the harvesting, while men are banned from ponds alongside the lake. During the bloom season in sum- entering the water, since it is a deep-rooted belief that they mer, when Arthrospira forms thick mats on the lake, people would make the lake barren. Arthrospira biomass is har- in boats collect a dense concentration of Arthrospira in buck- vested from Lake Kossorom throughout the year, with mini- ets (Fig. 25.18a ). This biomass is harvested on inclined mum yield in December and January, and a maximum from fi lters, washed with fresh water, dewatered and pressed again. June to September during the rainy season. The sun-dried The paste is then extruded into noodle-like fi laments which product is used by the people who harvest it, as well as being are dried in the sun on transparent plastic sheets (Fig. 25.18b ). sold on many local markets. It has been estimated that up to Dried chips are taken to a pharmaceutical factory in Yangoon, 4,000 t dried Arthrospira biomass are harvested every year pasteurized, and pressed into tablets (Habib et al. 2008 ) . A by local tribes in different countries from 14 wadis fl ooded more detailed study on the consumption patterns in these by Lake Chad during the rainy season. According to Sodelac various countries, as well as on the strains used, could A (2000 ) in 2000, over 90% of the production was sold by more detailed study on the consumption patterns, as well as female producers. The price of traditional dihé at production on the strains used, could provide valuable information for sites in 2009 ranged between US$ 0.678 and 1.1 kg−1 . the further development of the Arthrospira production, pro- A survey carried out among the Kanembu who harvest cessing and consumption industry. Arthrospira from Lake Kossorom in the Prefecture of Lac The accumulated knowledge of the ecology, physiology (Chad) has added considerable understanding of local eco- and biochemistry of Arthrospira has made its commercial nomics (Abdulqader et al. 2000) . The trading value of the production feasible. At present the photosynthetic ef fi ciency 25 Arthrospira (Spirulina) 701 of Arthrospira is still well below (2–3%) of the theoretical Baurain D, Renquin L, Grubisic S, Scheldeman P, Belay A, Wilmotte A maximum, which can be set at about 10% total solar light. (2002) Remarkable conservation of internally transcribed spacer sequences of Arthrospira (“Spirulina ”) (Cyanophyceae, Light saturation it has indicated to be one of the most important Cyanobacteria). Strains from four continents and of recent and factors which limit the full exploitation of the photosynthetic 30-years old samples from Africa. J Phycol 38:384–393 capacity. More work is required to provide strain selection Belay A (2002) The potential application of Spirulina (Arthrospira) as and better understanding of the factors governing growth, a nutritional and therapeutic supplement in health management. J Am Nutra Assoc (JANA) 5(2):27–48 especially light utilization in dense cultures, to support this Belay A (2008) Spirulina (Arthrospira ): production and quality assur- growing agro-industry. ance. In: Gershwin ME, Belay A (eds) Spirulina in human nutrition and health. CRC Press/Taylor & Francis Group, London, pp 1–25, 312 pp Acknowledgements The authors (TG and SC) thank the Italian Space Belkin S, Boussiba S (1991) Resistance of Spirulina platensis to ammo- Agency for fi nancial support through the MoMa project (contract number nia at high pH values. Plant Cell Physiol 32:953–958 I/014/06/0). Partial funding support was provided for joint projects in Bergman AN, Laurent P, Otiang’a-Oqwiti G, Bergman HL, Walsh PJ, the framework of Bilateral Scienti fi c Agreement (2010–2012) between the Wilson P, Wood CM (2003) Physiological adaptation of the gut in National Research Council of Italy and the Czech Academy of Science. the Lake Magadi tilapia, Alcolapia grahami, an alkaline-ad saline We thank Prof. Brian Whitton for his critical and helpful comments. adapted teleost fi sh. Comp Biochem Physiol A 136:701–715 Bernard Ch (1909) Sur quelques algues unicellulaires d’eau douce récoltés dans le Domaine Malais. Dep Agric Indes Néerlandaises, Buitzorg, 94 pp References Blumwald E, Tel-Or E (1982) Osmoregulation and cell composition in salt adaptation of Nostoc muscorum . Arch Microbiol 132:168–172 Abdulqader G, Barsanti L, Tredici M (2000) Harvest of Arthrospira Blumwald E, Mehlhorn RJ, Packer L (1984) Salt adaptation mecha- platensis form Lake Kossorom (Chad) and its household usage nisms in the cyanobacterium Synechococcus 6311. In: Sybesma S among the Kanembu. J Appl Phycol 12:493–498 (ed) Advances in photosynthesis research. Martinus Nijhoff/Dr W Abeliovich A, Azov Y (1976) Toxicity of ammonia to algae in sewage Junk Publs, The Hague, pp 627–630 oxidation ponds. Appl Environ Microbiol 31(6):801–806 Bocci F, Ferrari F, Materassi R, Mannelli D (1980) Prime ricerche sulla Aikawa S, Izumi Y, Matsuda F, Hasunuma T, Chang JS, Kondo A crescita di Spirulina platensis e Spirulina maxima in coltura fotolim- (2012) Synergystic enhancement of glycogen production in itata. In: Atti XIX Congr Naz Soc Ital Microbiol, Catania (Italy) Arthrospira platensis by optimization of light intensity and nitrate July 5–8 supply. Bioresour Technol 108: 211–215 Booker MJ, Walsby AE (1979) The relative form resistance of straight Alcocer J, Williams WD (1996) Historical and recent changes in Lake and helical blue-green algal fi laments. Br Phycol J 14:141–150 Texoco, a saline lake in Mexico. Int J Salt Lake Res 5:45–61 Borowitzka LJ (1986) Osmoregulation in blue-green algae. In: Round Aldea R, Guţu A, Nicoară A, Dragoş N (2002) Genetic variation of FE, Chapman DJ (eds) Progress in phycological research, vol 4. Arthrospira (Spirulina) strain assessed by PCR techniques based on Biopress Ltd, Bristol, pp 243–256 genomic repetitive sequences. Contributii Botanice 37:127–136 Bourrelly P (1970) Les Algues d’Eau Douce. III Les Algues bleues et An J, Carmichael WW (1996) Technical booklet for the microalgae bio- rouges, les Eugléniens, Peridiniens et Cryptomonadines. Editions N mass industry: detection of microcystins and nodularins using an Boubée & Cie, Paris, 512 pp enzyme linked immunosorbant assay (ELISA) and a protein phos- Boussiba S (1989) Ammonium uptake in the alkalophilic cyanobacte- phatase inhibition assay (PPIA). Department of Biological Sciences, rium Spirulina platensis . Plant Cell Physiol 32:303–314 Wayne State University, Dayton, OH Boussiba S, Gibson J (1991) Ammonia traslocation in cyanobacteria. Asada K (1994) Mechanisms for scavenging reactive molecules gener- FEMS Microbiol Lett 88:1–14 ated in chloroplasts under light stress. In: Baker NR, Bowyer JR Boussiba S, Affalo C (2005) An insight into the future of microalgal (eds) Photoinhibition of photosynthesis from molecular mecha- biotechnology. Innov Food Technol 28:37–39 nisms to the fi eld. Bios Scienti fi c Publishers, Oxford, pp 129–142 Brandily MY (1959) Depuis des lustres une tribu primitive du Tchad Averdano MC, Maeso ES, Nieva M, Fernandez Valiente E (1989) Effect exploite la nourriture de l’an 2000. Sci Avenir 152:516–519 of sodium de fi ciency on the ultrastructure of the cyanobacterium Busson F (1971) Spirulina platensis (Gom.) Geitler et Spirulina geitleri Anabaena PCC 7119. J Plant Physiol 135:409–415 J De Toni, Cyanophycées Alimentaires. Service de Santé, Marseille Balloni W, Tomaselli L, Giovannetti L, Margheri MC (1980) Biologia Campbell D, Hurry V, Clarke A, Gustafsson P, Oquist G (1998) fondamentale del genere Spirulina . In: Materassi R (ed) Prospettive Chlorophyll fl uorescence analysis of cyanobacterial photosynthesis della Coltura Massiva di Spirulina in Italia. CNR Rome, pp 49–85 and acclimation. Microbiol Mol Biol Rev 6:667–683 Ballot A, Krienitz L, Kotut K, Wiegand C, Metcalf JS, Codd GA, Carr NG, Whitton BA (eds) (1973) The biology of blue-green algae. Pfl ugmacher S (2004) Cyanobacteria and cyanobacterial toxins in Blackwell Scienti fi c Publications, Oxford, 676 pp three alkaline Rift Valley lakes of Kenya – Lakes Bogoria, Nakuru Carr NG, Whitton BA (eds) (1982) The biology of cyanobacteria. and Elmenteita. J Plankton Res 26:925–953 Blackwell Scienti fi c Publications, Oxford, 688 pp Ballot A, Krienitz L, Kotut K, Wiegand C, P fl ugmacher S (2005) Castenholz RW (1989) Subsection III, order oscillatoriales. In: Staley Cyanobacteria and cyanobacterial toxins in the alkaline crater lakes JT, Bryant MP, Pfennig N, Holt JG (eds) Bergey’s manual of sys- Sonachi and Simbi, Kenya. Harmful Algae 4:139–150 tematic bacteriology, vol 3. Williams and Wilkins Co, Baltimore, pp Ballot A, Kotut K, Novelo E, Krienitz L (2009) Changes of phytoplankton 1771–1780 communities in Lakes Naivasha and Oloidien, examples of degrada- Castenholz RW (2001) Form-genus I Arthrospira Stizenberger 1852. tion and salinization of lakes in the Kenyan Rift Valley. Hydrobiologia In: Boone DR, Castenholz RW, Garrity GM (eds) Bergey’s manual 632:359–363 of systematic bacteriology, vol 1, 2nd edn. Springer, New York, pp Bao Y, Liu M, Wu X, Cong W, Ning Z (2012) In situ carbon supple- 542–543 mentation in large-scale cultivations of Spirulina platensis in open Ciferri O (1983) Spirulina , the edible microorganism. Microbiol Rev raceway pond. Biotechnol Bioprocess Eng 17:93–99 47:551–578 702 C. Sili et al.

Clément G (1968) A new type of food algae. In: Mateles RJ, Tannenbaum Fuzinato S, Fodora A, Subakov-Simic G (2010) Arthrospira fusiformis SR (eds) Single-cell protein. Massachusetts Institute of Technology (Voronichin) Komárek et Lund (Cyanoprocariota)- A new species Press, Cambridge, MA for Europe. Algol Stud 134:17–24 Coe MJ (1966) The biology of Tilapia grahami Boulenger in Lake Gallegos AJ (1993) The past, present and future of algae in Mexico. Magadi, Kenya. Acta Trop 23:146–177 Bull Inst Océanograph Monaco 12:133–139 Colwell RR (1970) Polyphasic taxonomy of the genus Vibrio : numeri- Gao K, Li P, Watanabe T, Helbling EW (2008) Combined effects of cal taxonomy of Vibrio cholerae , Vibrio parahaemolyticus , and ultraviolet radiation and temperature on morphology, photosynthe- related Vibrio species. J Bacteriol 104:410–433 sis, and DNA of Arthrospira ( Spirulina ) platensis (Cyanophyta). J Compère P (1974) Cyanophycées de la région du lac Tchad, taxons, Phycol 44:777–786 combinaisons et noms nouveaux. Bull Jard Bot Nat Belg 44: Gardner NL (1917) New Pacifi c coast marine algae I. Univ Calif Publ 17–21 Bot 6:377–416, Plates 31–35 Dadheech PK, Ballot A, Casper P, Kotut K, Novelo E, Lemma B, Geitler L (1925) Cyanophyceae. In: Pascher Süsswasserfl ora 12. G Pröschold T, Krienitz L (2010) Phylogenetic relationship and diver- Fisher-Verlag, Jena, 450 pp gence among planktonic strains of Arthrospira (Oscillatoriales, Geitler L (1932) Cyanophyceae. In: Rabenhorst’s Krytogamen-Flora Cyanobacteria) of African, Asian and America origin deduced by von Deutschland, Österreich und der Schweiz 14. Akademische 16S-23S ITS and phycoyanin operon sequences. Phycologia Verlagsgesellschaft, Leipzig, 1196 pp 49(4):361–372 Gershwin ME, Belay A (2008) In: Gershwin ME, Belay A (eds) Dainippon Ink & Chemicals Inc. (1980) Production of highly puri fi ed Spirulina in human nutrition and health. CRC Press/Taylor & alcoholophilic phycocyanin. Japanese Patent 80 77, 890 Francis Group, Boca Raton Dainippon Ink & Chemicals Inc. (1981) Cosmetics containing water Gomont M (1892) Monographie des Oscillariées. Ann Sci Nat Bot, Sér soluble phycocyanin. Japanese Patent 7 9–138755 7, 15:263–368; 16:91–264 Dangeard P (1940) Sur une algue bleue alimentaire pour l’homme: Gong H, Tang Y, Wang J, Wen X, Zhang L, Lu C (2008) Characterization Arthrospira platensis (Nordst.) Gomont. Act Soc Linn Bordx of photosystem II in salt stressed cyanobacterial Spirulina platensis 91:39–41 cells. Biochim Biophys Acta 1777:488–495 De Toni GB (1935) Noterelle di nomenclatura algologica 3:1–2 Grant WD (2006) Alkaline Environments and Biodiversity. In: Gerday Desikachary TV (1959) Cyanophyta. Indian Council of Agricultural C, Glansdorff N (eds) Extremophiles. In Encyclopedia of Life Research, New Delhi, pp 187–198, 686 pp Support Systems (EOLSS). Eolss Publishers, Oxford. htpp://www. Desikachary TV, Jeeji Bai N (1992) Taxonomic studies in Spirulina . In: eolss.net Seshradi CV, Jeeji Bai N (eds) Spirulina ETTA national symposium. Grant WD, Mwatha WE, Jones BE (1990) Alkaliphiles: ecology, diver- MCRC, Madras, pp 12–21 sity and applications. FEMS Microbiol Rev 75:255–270 Desikachary TV, Jeeji Bai N (1996) Taxonomic studies in Spirulina II. Greque de Morais M, Da Cruze Reichert C, Dalcaton F (2008) Isolation The identi fi cation of Arthrospira (“ Spirulina ”) strains and natural and characterization of a new Arthrospira strain. Z Naturforsch samples of different geographical origins. Arch Hydrobiol Suppl 63c:144–150 116 Algol Stud 83:163–168 Guglielmi G, Cohen-Bazire G (1982) Structure et distribution des pores Ehrenfeld J, Cousin JL (1984) Ionic regulation of the unicellular green et des perforations de l’enveloppe de peptidoglycane chez quelques alga Dunaliella tertiolecta: response to hypertonic shock. J Membr cyanobactéries. Protistologica 18:151–165 Biol 77:45–55 Guterman H, Vonshak A, Ben-Yaakov S (1989) Automatic on-line El-Bestawy E, Bellinger EG, Sigee DC (1996) Elemental composition growth estimation method for outdoor algal biomass production. of phytoplankton in a subtropical lake: X-ray microanalytical stud- Biotechnol Bioeng 34:143–152 ies on the dominant algae Spirulina platensis (Cyanophyta) and Habib B, Parvin, Huntington TC, Hasan MR (2008) A review on cul- Cyclotella meneghiniana (Bacillariophyceae). Eur J Phycol ture, production and use of Spirulina as food for humans and feeds 31:157–166 for domestic animal and fi sh. FAO Fisheries and Aquaculture Espie GS, Miller AG, Canvin DT (1988) Characterization of the Na + - Circular No. 1034, ISBN 978-92-5-106-106-0 Requirement in cyanobacterial photosynthesis. Plant Physiol Hindák F (1985) Morphology of trichomes in Spirulina fusiformis 88:757–763 Voronichin from Lake Bogoria, Kenya. Arch Hydrobiol Suppl 71 Fathi AA, Abdelzaher A, Flower RJ, Ramdani M, Kraïem MM (2001) Algol Stud 38(39):209–218 Phytoplankton communities of North African wetland lakes: the Hongsthong A, Sirijuntarut M, Prommeenate P, Thammathorn S, CASSARINA Project. Aquatic Ecology 35:303–318 Bunnag B, Cheevadhanarak S, Tanticharoen M (2007) Revealing Ferreira LS, Rodrigues MS, Converti A, Sato S, Carvalho JCM (2012) differentially expressed proteins in two morphological forms of Arthrospira (Spirulina) platensis cultivation in tubular photobiore- Spirulina platensis by proteomic analysis. Mol Biotechnol

actor: use of no-cost CO2 from ethanol fermentation. Appl Energy 36:123–130 92:379–385 Horikoshi K, Akiba T (1982) Alkalophilic microorganisms. Springer, Fogg GE, Stewart WDP, Fay P, Walsby AE (1973) The blue-green Berlin algae. Academic, London, 459 pp Iltis A (1968) Tolérance de salinité de Spirulina platensis (Gom.) Fott B, Karim AGA (1973) Spirulina plankton community in a lake in Geitler, (Cyanophyta ) dans les mares natronées du Kanem (Tchad). Jebel Marra, Sudan. Arch Protistenk 115:408–418 Cah ORSTOM, sér Hydrobiol 2:119–125 Fox DL (1976) Animal biochromes and structural colours, 2nd edn. Iltis A (1969a) Phytoplancton des eaux natronées du Kanem (Tchad). I. University of California Press, Berkeley, p 433 Les lacs permanents a Spirulines. Cah ORSTOM, sér Hydrobiol Fox RD (1985) Spirulina, the alga that can end malnutrition. Futurist 3:29–44 19:30–35 Iltis A (1969b) Phytoplancton des eaux natronées du Kanem (Tchad). Fox RD (1996) Spirulina production and potential. Edisud, Aix-en- II. Les mares temporaires. Cah ORSTOM, sér Hydrobiol 3:3–19 Provence Iltis A (1970) Phytoplancton des eaux natronées du Kanem (Tchad). III. Fry IV, Hufl ejt M, Erber WWA, Peshek GA, Packer L (1986) The role Variations annuelles du plancton d’une mare temporaire. Cah of respiration during adaptation of the freshwater cyanobacterium ORSTOM, sér Hydrobiol 4:53–59 Synechococcus 6311 to salinity. Arch Biochem Biophys 244: Iltis A (1971) Phytoplancton des eaux natronées du Kanem (Tchad). V. 686–690 Les lacs mésohalins. Cah ORSTOM, sér Hydrobiol 5:73–84 25 Arthrospira (Spirulina) 703

Iltis A (1972) Algues des eaux natroné du Kanem (Tchad). Primo. salinity is characterised by two distinct phases. Aust J Plant Physiol Premiere saltie. Cah ORSTOM, sér Hydrobiol 4:129–134 26:283–292 Iltis A, Riou-Duwat S (1971) Variations saisonnières du peuplement en Lu Y-M, Xiang W-Z, Wen Y-H (2011) Spirulina ( Arthrospira ) industry rotifères des eaux natronées du Kanem (Tchad). Cah ORSTOM, sér in Inner Mongolia of China: current status and prospects. J Appl Hydrobiol 5:101–112 Phycol 23:264–269 Jeeji Bai N (1985) Competitive exclusion or morphological transforma- Ma Z, Gao K (2009) Photoregulation of morphological structure and its tion? A case study with Spirulina fusiformis . Arch Hydrobiol Suppl physiological relevance in the cyanobacterium Arthrospira 71 Algol Stud 38(39):191–199 ( Spirulina ) platensis . Planta 230:329–337 Jeeji Bai N (1999) A taxonomic appraisal of the genera Spirulina and Maglione G. (1969) Premières données sur le regime hydrogéochimique Arthrospira . In: Mahato AK, Vidyavati (eds) Recent trends in algal des lacs permanents du Kanem (Tchad). Cah ORSTOM, sér taxonomy, vol I, Taxonomical issues. APC Publications PVT LTD, Hydrobiol 3:121–141 New Delhi, pp 253–272 Manen JF, Falquet J (2002) The cpcB-cpcA locus as a tool for the Jeeji Bai N, Seshadri CV (1980) On coiling and uncoiling of trichomes in genetic characterization of the genus Arthrospira (Cyanobacteria): the genus Spirulina . Arch Hydrobiol Suppl 60 Algol Stud 26:32–47 evidence for horizontal transfer. Int J Syst Evol Microbiol Kebede E, Ahlgren G (1996) Optimum growth conditions and light 52:861–867 utilisation ef fi ciency of Spirulina platensis (equal Arthrospira fusi- Margheri MC, Tomaselli Feroci L, Materassi R (1975) Contributo sper- formis ) (Cyanophyta) from Lake Chitu, Ethiopia. Hydrobiologia imentale alla sistematica del genere Spirulina Turpin. In: Atti XVII 332:99–109 Cong Naz Soc Ital Microbiol, Padova, vol II, pp 891–901 Kerfeld CA (2004) Structure and function of the water-soluble carote- Marquez FJ, Sasaki K, Nishio N, Nagai S (1995) Inhibitory effect of noid-binding proteins of cyanobacteria. Photosynth Res oxygen accumulation on growth of Spirulina platensis . Biotechnol 81:215–225 Lett 17:222–228 Kirilovsky D (2007) Photoprotection in cyanobacteria: the orange caro- Martel A, Yu S, Garcia-Reina G, Lindblad P, Pedersén M (1992) tenoid protein (OCP)-related non-photochemical-quenching mech- Osmotic-adjustment in the cyanobacterium Spirulina platensis : anism. Photosynth Res 93:7–16 presence of an a -glucosidase. Plant Physiol Biochem 30:573–578 Komárek J, Anagnostidis K (2005) Cyanoprokaryota – Part 2: oscilla- Melack JM (1979) Photosynthesis and growth of Spirulina platensis toriales. In: Büdel B, Krienits L, Gärtner G, Schagerl M (eds) (Cyanophyta). In an equatorial lake (Lake Simbi, Kenya). Limnol Süsswasserfl ora von Mitteleuropa 19/2. Elsevier/Spektrum, Oceanogr 24:753–760 Heidelberg, pp 341–353, 759 pp Miller AG, Turpin DH, Canvin DT (1984) Na + Requirement for growth, Komárek J, Hauer T (2011) CyanoDB.cz – On-line database of photosynthesis, and pH regulation in the alkalotolerant cyanobacte- cyanobacterial genera. – Word-wide electronic publication, rium Synechococcus leopoliensis . J Bacteriol 159:100–106 University of South Bohemia & Institute of Botany AS CR. http:// Molitor V, Erber W, Peschek GA (1986) Increased levels of cytochrome www.cyanodb.cz oxidase and sodium proton antiporter in the plasma membrane of Komárek J, Lund JWG (1990) What is “Spirulina platensis ” in fact? Anacystis nidulans after growth in sodium enriched media. FEBS Arch Hydrobiol Suppl 85 Algol Stud 58:1–6 Lett 204:251–254 Krause GH (1994) The role of oxygen in photoinhibition of photosyn- Mühling M, Harris N, Belay A, Whitton BA (2003) Reversal of helix thesis. In: Foyer CH, Mullinex PM (eds) Cause of photooxidative orientation in the cyanobacterium Artrhospira . J Phycol 39:360–367 stress and amelioration of defences systems in plants. CRC Press, Mühling M, Somer fi eld PJ, Harris N, Belay A, Whitton BA (2006) Boca Raton, pp 42–76 Phenotypic analysis of Arthrospira (Spirulina) strains (cyanobacte- Laliberté G, Olguín EJ, de la Noüe J (1997) Mass cultivation and waste- ria). Phycologia 45(2):148–157 water treatment using Spirulina. In: Vonshak A (ed) Spirulina plat- Murata N, Takahashi S, Nishiyama Y, Allakhverdiev SI (2007) ensis (Arthrospira ): physiology, cell-biology and biotechnology. Photoinhibition of PSII under environmental stress. Biochim Taylor & Francis Ltd, London, pp 159–173, 233 pp Biophys Acta:1767:414–421 Lee YK (1997) Commercial production of microalgae in the Asia- Mussagy A, Wilmotte A, Cronberg G (2006) The cyanophyte Paci fi c rim. J Appl Phycol 9:403–4511 Arthrospira fusiformis from Mozambique, Africa: morphological Léonard J, Compère P (1967) Spirulina platensis (Gom.) Geitler, algue and molecular characterization. Algol Stud 121:59–73 bleue de grande valeur alimentaire par sa richesse en protéines. Bull Noor P, Akhtar N, Munshi JL, Begum S (2008) Spirulina culture in Jard Bot Nat Belg 37:1–23 Bangladesh XII. Effects of different culture media, different culture Leverenz JW, Falk S, Pilström CM, Samuelson G (1990) The effect of vessels and different cultural conditions on coiled and straight on the photosynthetic light-response curve of green plant cells fi lament characteristics of Spirulina . Bangladesh J Sci Ind Res ( Chlamydomonas reinhardtii ). Planta 182:161–168 43(3):369–376 (Available at www.banglajol.info) Lewin RA (1980) Uncoiled variants of Spirulina platensis Oberholster PJ, Botha AM, Ashton PJ (2009) Appearance of new taxa: (Cyanophyceae: Oscillatoriaceae). Arch Hydrobiol Suppl 60 Algol invertebrates phytoplankton and bacteria in a alkaline, saline, mete- Stud 26:48–52 orite crater lake, South Africa. Fundam Appl Limnol Archiv Li BS, Qiao V, Tian XY (2003) Potential to development Spirulina plat- Hydrobiol 173. doi: 10.1127/1863-9135/2009

ensis with alkaline lakes in Orodos Plateau. Plant Mag 06:18–20 Ogawa T, Aiba S (1978) CO2 assimilation and growth of a blue-green (cited by Lu et al. 2011) alga, Spirulina platensis , in continuous culture. J Appl Chem Lincoln EP, Hall TW, Koop B (1983) Zooplankton control in mass algal Biotechnol 28:5151–5157 cultures. Aquaculture 32:331–337 Ohmori K, Ehira S, Kimura S, Ohmori M (2009) Changes in the amount Long SP, Humphries S, Falkowski PG (1994) Photoinhibition of photo- of cellular trehalose, the activity of maltooligosyl trehalose synthesis in nature. Annu Rev Plant Physiol Plant Mol Biol hydrolase, and the expression of its gene in response to salt stress 45:633–662 in the cyanobacterium Spirulina platensis . Microbes Environ Lu C, Vonshak A (2002) Effects of salinity on photosystem II function 24(1):52–56 in cyanobacterial Spirulina platensis cells. Physiol Plant Okoth OE, Mucai M, Shivoga WA, Miller SN, Rasowo J, Ngugi CC 114:405–413 (2009) Spatial and seasonal variations in phytoplankton community Lu C, Torzillo G, Vonshak A (1999) Kinetic response of photosystem II structure in alkaline-saline Lake Nakuru, Kenya. Lake & Reservoirs. photochemistry in the cyanobacterium Spirulina platensis to high Res Manag 14:57–69 704 C. Sili et al.

Padan E, Zilberstein D, Schuldiner S (1981) pH Homeostasis in bacte- Spiller S, Denbeaux G, Jones G, Pearson AL (2000) Fine structure of ria. Biochim Biophys Acta 650:151–166 cyanobacteria, Spirulina platensis and Spirulina subsalsa , as viewed Padisák J, Soróczki-Pintér E, Rezner Z (2003) Sinking properties of by x-ray microscope, XM-1, beamline 6.1.2. In Compendium 2000. some phytoplankton shapes and the relation of form resistance to The Advanced Light Source (ALS), Lawrence Berkeley National morphological diversity of plankton – an experimental study. Laboratories, General ALS Publications Hydrobiologia 500:243–257 Stizenberger E (1852) Spirulina und Arthrospira (nov. gen.). Hedwigia Palinska KA, Krumbein WE (2000) Perforation patterns in the peptido- 1:32–41 glycan wall of fi lamentous cyanobacteria. J Phycol 36:139–145 Talling JF, Talling IB (1965) The chemical composition of African lake Peschek GA, Obinger C, Fromwald S, Bergman B (1994) Correlation waters. Int Rev Hydrobiol 50:1–32 between immuno-gold labels and activities of cytochrome-c oxidase Thein M (1993) Production of Spirulina in Myanmar. Bull Inst (aa-type) in membranes and salt stressed cyanobacteria. FEMS Océanograph Monaco 12:175–178 Microbiol Lett 124:431–438 Tomaselli L (1997) Systematic and ecology. In: Vonshak A (ed) Pogoryelov D, Sudhir P-R, Kovács L, Gombos Z, Brown I, Garab G Spirulina platensis (Arthrospira ): physiology, cell-biology and bio- (2003) Sodium dependency of the photosynthetic electron transport technology. Taylor & Francis Ltd, London, pp 1–19, 233 pp in the alkaliphilic cyanobacterium Arthrospira platensis . J Bioenerg Tomaselli L, Giovannetti L, Margheri MC (1981) On the mechanism of Biomembr 35(5):427–437 trichome breakage in Spirulina platensis and S. maxima . Ann Qiao C, Li BS, Zeng ZQ (2001) Alkaline lakes and Spirulina Microbiol 31:27–31 ( Arthrospira ) resources in sandy land of Erdos. J Arid Land Res Tomaselli L, Torzillo G, Giovannetti L, Pushparaj B, Bocci F, Tredici Environ 15(4):86–91 M, Papuzzo T, Balloni W, Materassi R (1987) Recent research on Ramus J (1981) The capture and transduction of light energy. In: Spirulina in Italy. Hydrobiologia 151(152):79–82 Lobban CS, Wynne MJ (eds) The biology of seaweeds, botanical Tomaselli L, Giovannetti L, Sacchi A, Bocci F (1988) Effects of tem- monographs, vol 17. Blackwell Scientifi c Publications, Oxford, pp perature on growth and biochemical composition in Spirulina plat- 458–492 ensis strain M2. In: Staedler T, Mollion J, Verdus MC, Karamanos Reed RH, Richardson DL, Stewart WDP (1985) Na+ uptake and extru- Y, Morvan H, Christiaen D (eds) Algal biotechnology. Elsevier sion in the cyanobacterium Synechocystis PCC 6714 in response to Applied Sciences, London/New York, pp 305–314 hypersaline treatment. Evidence for transient changes in plasma- Tomaselli L, Giovannetti L, Torzillo G (1993) Physiology of stress lemma Na+ permeability. Biochim Biophys Acta 814:347–353 response in Spirulina spp. Bull Inst Océanograph, Monaco 12:65–75 Rich F (1931) Notes on Arthrospira platensis . Rev Algol 6:75–82 Tomaselli L, Palandri MR, Tredici MR (1996) On the correct use of the Rich F (1932) Report on the Percy Sladen Expedition to some Rift Spirulina designation. Arch Hydrobiol Suppl 116 Algol Stud Valley Lakes in Kenya in 1929. IV. Phytoplankton from the Rift 83:539–548 Valley Lakes in Kenya. Ann Mag Nat Hist, ser.10, 10:233–262 Thomasson K (1960) Ett fall av tropisk vattenblomming. [A case of Richmond A (2004a) In: Richmond A (ed) Handbook of microalgal tropical waterbloom] Bot Notiser 113(2):214–216 culture: biotechnology and applied phycology. Blackwell Science Torzillo G, Vonshak A (1994) Effect of light and temperature on the Ltd, Oxford, 566 pp photosynthetic activity of the cyanobacterium Spirulina platensis . Richmond A (2004b) Biological principles of mass cultivation. In: Biomass Bioenergy 6:399–404 Richmond A (ed) Handbook of microalgal culture: biotechnology Torzillo G, Vonshak A (2003) Biotechnology of algal mass cultivation. and applied phycology. Blackwell Science Ltd, Oxford, pp In: Fingerman M, Nagabhushanam R (eds) Recent advances in 125–177 marine biotechnology, vol 9, Biomaterials and bioprocessing. Rippka R, Herdman M (1992) Catalogue of strains. Pasteur culture col- Science Publishers, Inc, Plymouth, pp 45–77, 385 pp lection of cyanobacterial strains in axenic culture. Institut Pasteur, Torzillo G, Giovannetti L, Bocci F, Materassi R (1984) Effect of oxygen Paris, 103 pp concentration on the protein content of Spirulina biomass. Rodrigues MS, Ferreira LS, Converti A, Sato S, Monteiro de Carvalho Biotechnol Bioeng 26:1134–1135 JC (2011) Infl uence of ammonium sulphate feeding time on the fed- Torzillo G, Pushparaj B, Bocci F, Balloni W, Materassi R, Florenzano batch Arthrospira (Spirulina) platensis cultivation and biomass G (1986) Production of Spirulina biomass in closed photobioreac- composition with and without pH control. Bioresour Technol tors. Biomass 11:61–74 102:6587–6592 Torzillo G, Sacchi A, Materassi R, Richmond A (1991) Effect of tem- Scheldeman P, Baurain D, Bouhy R, Scott M, Mühling M, Whitton BA, perature on yield and night biomass loss in Spirulina platensis Belay A, Wilmotte A (1999) Arthrospira (‘Spirulina ’) strains from grown outdoors in tubular photobioreactors. J Appl Phycol four continents are resolved into two only two clusters, based on 3:103–108 ARDRA (Amplifi ed Ribosomal DNA Restriction Analysis) of the Torzillo G, Bernardini P, Masojidek J (1998) On-line monitoring of ITS (Internal Transcribed Spacer). FEMS Microbiol Lett chlorophyll fl uorescence to assess the extent of photoinhibition of 172:213–222 photosynthesis induced by high oxygen concentration and low tem- Schlesinger P, Belkin S, Boussiba S (1996) Sodium deprivation under perature and its effect on the productivity of outdoor cultures of alkaline conditions causes rapid death of the fi lamentous cyanobac- Spirulina platensis (Cyanobacteria). J Phycol 34:504–510 terium Spirulina platensis . J Phycol 32:608–613 Torzillo G, Komenda J, Kopecky J, Faraloni C, Masojidek J (2003a) Shimamatsu H (2004) Mass production of Spirulina , an edible Photoinhibitory stress induced by high oxygen and low temperature microalga. Hydrobiologia 512:39–44 in outdoor cultures of Arthrospira platensis grown in closed photo- Sili C, Abdulqader G, Tredici MR (1999) Photosynthetic biocenosis of bioreactors. In: Abstracts of third European phycological congress, two soda lakes of the north-east fringe of Lake Chad. In Abstracts of Belfast, Ireland, July 21–26 the 8th international conference of applied algology “Algae and Torzillo G, Pushparaj B, Masojidek J, Vonshak A (2003b) Biological human affairs in the 21st century”, Montecatini Terme, p 318 constraints in algal biotechnology. Biotechnol Bioprocess Eng Sodelac (2000) Etude de préfaisabilité du developpement de la produc- 8:338–348 tion de spirulines. Répubblique du Tchad. Ministére de l’Agriculture. Tredici MR, Papuzzo T, Tomaselli L (1986) Outdoor mass culture of Tractebel Consult (Belgique). Financement: Fond African de Spirulina maxima in sea-water. Appl Microbiol Biotechnol Dèveloppement. Contract N 003/SODELA C/99 24:42–50 25 Arthrospira (Spirulina) 705

Tsen CT, Chang TP (1990) Ultrastrukturen von vier “Spirulina”-Arten Vonshak A, Abeliovich A, Boussiba S, Arad S, Richmond A (1982) (Cyanophyceae). Arch Hydrobiol Suppl 87 Algol Stud 60:33–41 Production of Spirulina biomass: effects of environmental factors Tuite CH (1981) Standing crop densities and distribution of Spirulina and population density. Biomass 2:175–185 and benthic diatoms in East African alkaline saline lakes. Freshw Vonshak A, Boussiba S, Abeliovich A, Richmond A (1983) Production Biol 11:345–360 of Spirulina biomass: maintenance of monoalgal culture. Biotechnol Turpin PJF (1829) Spirulina oscillarioide. In: Dictionnaire des Sciences Bioeng 25:341–351 Naturelles, De Lévrault, Paris vol. 50, pp 309–310 Vonshak A, Guy R, Guy M (1988a) The response of the fi lamentous Van Eykelenburg C (1977) On the morphology and ultrastructure of the cyanobacterium Spirulina platensis to salt stress. Arch Microbiol cell wall of Spirulina platensis. Antonie Van Leeuwenhoek J 150:417–420 Microbiol 43:89–94 Vonshak A, Guy R, Poplawsky R, Ohad I (1988b) Photoinhibition and Van Eykelenburg C (1979) The ultrastructure of Spirulina platensis in its recovery in two different strains of Spirulina . Plant Cell Physiol relation to temperature and light intensity. Antonie Van Leeuwenhoek 29:721–726 J Microbiol 45:369–375 Vonshak A, Chanawongse L, Bunnang B, Tanticharoen M (1996a) Van Eykelenburg C, Fuchs A (1980) Rapid reversible macromorphologi- Light acclimation and photoinhibition in three Spirulina platensis cal changes in Spirulina platensis . Naturwissenschaften 67:200–204 (cyanobacteria) isolates. J Appl Phycol 8:35–40 Van Liere L, Mur L (1979) Growth kinetics of Oscillatoria agardhii Vonshak A, Torzillo G, Accolla P, Tomaselli L (1996b) Light and oxy- Gomont in continuous culture limited in its growth by the light gen stress in Spirulina platensis (cyanobacteria) grown outdoors in energy supply. J Gen Microbiol 115:153–159 tubular reactors. Physiol Plant 97:175–179 Vareschi E (1982) The ecology of lake Nakuru (Kenya). III: abiotic fac- Voronichin NN (1934) Biologie des bassins mineral des steppes tors and primary production. Oecologia 55:81–101 Koulondine. Expedition de l’Academie des Sciences d’URSS Verma K, Mohanty P (2000) Alterations in the structure of phycobili- 1931–1933 I. Leningrad. Trav Soviet pour serv l’étude ressour somes of the cyanobacterium Spirulina platensis in response to natur. Sér Siberie 8:182–183 enhanced Na + level. World J Microbiol Biotechnol 16:795–798 Wang ZP, Zhao Y (2005) Morphological reversion of Arthrospira Viti C, Ventura S, Lotti F, Capolino E, Tomaselli L, Giovannetti L ( Spirulina ) platensis (Cyanophyta): from linear to helical. J Phycol (1997) Genotypic diversity and typing of cyanobacterial strains of 41:622–628 the genus Arthrospira by very sensitive total DNA restriction pro fi le Welsh H (1965) A contribution to our knowledge of the blue-green analysis. Res Microbiol 148:605–611 algae of South West Africa and Bechuanaland. Nova Hedwigia 9 Vonshak A (1997a) Spirulina platensis (Arthrospira): physiology, cell- (1–4):131–162 biology and biotechnology. Taylor & Francis Ltd, London, 233 pp Wilson A, Ajilani G, Verbavatz J-M, Vass I, Kerfeld C, Kirilovsky D Vonshak A (1997b) Spirulina : growth, physiology and biochemistry. In: (2006) A soluble carotenoid protein involved in phycobilisome- Vonshak A (ed) Spirulina platensis ( Arthrospira): physiology, cell- related energy dissipation in cyanobacteria. Plant Cell 18:992–1007 biology and biotechnology. Taylor & Francis Ltd, London, pp 43–65 Wittrock VB, Nordstedt CFO (1884) Algae aquae dulcia exiccata. Fasc Vonshak A, Guy R (1992) Photoadaptation, photoinhibition and pro- 14, 679:59 ductivity in outdoor grown Spirulina platensis strains. Plant Cell Wu H, Gao K, Villafaňe VE, Watanabe T, Helbling EW (2005) Effects Environ 15:613–616 of solar UV radiation on morphology and photosynthesis of Vonshak A, Novoplansky N (2008) Acclimation to low temperature of fi lamentous cyanobacterium Arthrospira platensis . Appl Environ two Arthrospira platensis (cyanobacteria) strains involves down- Microbiol 71(9):5004–5013 regulation of PSII and improved resistance to photoinhibition. J Zarrouk C (1966) Contribution à l’étude d’une cyanophycée. In fl uence Phycol 44:1071–1079 de divers facteurs physiques et chimiques sur la croissance et la pho- Vonshak A, Richmond A (1988) Mass production of the blue-green tosynthèse de Spirulina maxima (Setchell et Gardner) Geitler. Ph D alga Spirulina : an overview. Biomass 15:233–247 thesis, University of Paris, France Vonshak A, Richmond A (1981) Photosynthetic and respiratory activity Zeng MT, Vonshak A (1998) Adaptation of Spirulina platensis to salin- in Anacystis nidulans adapted to osmotic stress. Plant Physiol ity-stress. Comp Biochem Physiol A 120:113–118 68:504–509 Zhao J, Briand JJ (1988) Sequential events in the photoinhibition of Vonshak A, Tomaselli L (2000) Arthrospira ( Spirulina ) Systematics Synechocystis under sodium stress. Plant Physiol 91:91–100 and ecophysiology. In: Whitton BA, Potts M (eds) The ecology of Zheng XY, Zhang MG, Dong YH, Gao ZH, Xu C, Han ZM, Zhang BZ, cyanobacteria. Their diversity in time and space. Kluwer Academic Sun DP, Wang KJ (1992) Salt lakes in Inner Mongolia. Science Publishers, Dordrecht, pp 505–522, 669 pp press, Beijing, pp 41–266 (in Chinese)