Spirulina, the Edible Microorganism

MICROBIOLOGICAL REVIEWS, Dec. 1983, p. 551-578 Vol. 47, No. 40146-0749/83/040551-28$02.00/0Copyright © 1983, American Society for Microbiology

Spirulina, the Edible MicroorganismORIO CIFERRI

Department of Genetics and Microbiology, University of Pavia, 27100 Pav'ia, Italy

INTRODUCTION............................................................. 551OCCURRENCE AND ISOLATION ................ ................................ 552MORPHOLOGY AND TAXONOMY............................................... 558PHYSIOLOGY ............................................................. 563BIOCHEMISTRY............................................................. 563CHEMICAL COMPOSITION..................................................... 566PRODUCTION ............................................................. 567NUTRITION AND TOXICOLOGY ................ ................................ 569CONCLUSIONS AND PROSPECTS OF THE UTILIZATION OF SPIRULINAAS A FOOD SOURCE ......................................................... 572

ACKNOWLEDGMENTS ......................................................... 574LITERATURE CITED ........................................................... 574

INTRODUCTIONIn 1940 the French phycologist Dangeard

described in a communication to the LinneanSociety of Bordeaux a sample received from Mr.Crdach, pharmacist with the French Colonialtroops stationed at Fort Lamy, at that time inFrench Equatorial Africa and now in the Repub-lic of Chad (31). The sample was obtained fromthe market of Massakory, a small village locatedapproximately 50 km east of Lake Chad. Dan-geard reported that the material called Dihd (ordid) in the local language (Kanenbou), was eatenby the native population, and was obtained asfollows: mats of microscopic algae, floating onthe surface of small lakes or ponds around LakeChad, were collected and sun dried on the sandyshores (Fig. 1). The hardened cakes were brokeninto small pieces and, without any further treat-ment, represented Dihe, object of some com-merce in the local markets. According to Mr.Crdach, Dihd was used to make sauces accom-panying the standard millet meal. On studyingthe samples of Dihd, Dangeard concluded that itwas "a true puree of a filamentous, spiral-shaped blue alga." The alga was Arthrospira (=Spirulina) platensis. A colleague of Dangeard,the abbot Fremy, informed him that the orga-nism had already been identified by Rich as themain constituent of the phytoplankton in a num-ber of lakes in the Rift Valley of East Africa(128). Rich had also reported that the organismrepresented the main food source for the popula-tion of lesser flamingoes (Phoenicoptera) inhab-iting those lakes. Because of the war and, possi-bly, the limited circulation of the journal inwhich the communication was published, Dan-geard's report went unnoticed. Almost 25 yearslater, J. Leonard (a botanist participating in theBelgian Trans-Saharan expedition), while

looking for plant products in the native marketsin and around Fort Lamy, was struck by a'curious substance green bluish, sold as driedbiscuits" (91). Ldonard had rediscovered Diheand confirmed that it was composed almostexclusively of dried mats of S. platensis collect-ed from the waters of the alkaline lakes in thesubdesert Kanem area, northeast of Lake Chad.Ldonard and his colleague Compdre confirmedthe report by Dangeard that Dihe was consumedby the local populations and performed a firstgroup of chemical analyses that revealed a veryhigh protein content, close to 50% of the dryweight. Although impressive, this figure was anunderestimate since independent studies, car-ried out by the Institut Francaise du Pdtrole onlaboratory-grown S. platensis, gave protein con-tents that ranged from 62 to 68% of the dryweight (28).At the same time, the French group had begun

to investigate also another species of Spiriilina,S. maxima (= S. geitleri), that was growingabundantly in Lake Texcoco, near Mexico City(27, 40). Although there was no indication thatS. maxima was used in Mexico as a food, asearch of the historical literature revealed that,at the times of the Spanish conquest, S. maximawas harvested from the lake, dried, and sold forhuman consumption. The very attentive Spanishinvaders duly recorded all animals, plants, andfoods that they encountered in the newly con-quered territories. (Indeed, for instance, Colum-bus himself noted that, during his first visit toCuba in 1492, he saw a "type of grain likemillet" that the natives called maize.) BernalDiaz del Castillo, a member of Cortez' troops,described among the many astonishing itemsthat he saw in the market of Tenochtitlan (to-day's Mexico City) " . . . small cakes made

551

552 CIFERRI

FIG. 1. Preparation and sale of Dihe. (A) Sun-drying of S. platensis mats on the shores of LakeRombou (Republic of Chad), ca. 1967. (Photo by A.Iltis.) (B) DihE on sale in the village market of Massa-kory (Republic of Chad), ca. 1967. (Photo by A. Iltis.)

from a sort of a ooze which they get out of thegreat lake, and from which they make a breadhaving a flavour something like cheese" (36). Afew years later, a Franciscan friar, Bernardinoda Sahagiin, described how fishermen ...

with very fine nets in certain periods of the yearcollect a soft thing that is created on the watersof the lagoons of Mexico, and which curdles,and it is not grass nor earth, rather like hay . . .

of clear blue color, from which they make bread,that they eat cooked" (34) (Fig. 2). The nativescalled it Tecuitlatl, literally "stone's excre-ment" since, as Farrar has pointed out (42),"breeding" of minerals was still a common

belief in the 16th century. Tecuitlatl was men-tioned by historians of the conquest or visitingnaturalists up to the end of the 16th century.After that period, Tecuitlatl is not mentionedany longer, probably because the practice ofmaking Tecuitlatl disappeared soon after theconquest. It is possible that the local population,decimated also by repeated outbreaks of conta-gious diseases, could satisfy its alimentary needswith more conventional foods. Further, it ispossible that due to the profound social, politi-cal, and religious changes caused by the Spanishconquest many traditions were quickly lost (42).

Thus, over the ages, two populations, approx-imately 10,000 km apart, discovered indepen-dently and exploited the nutritional properties ofSpirulina. Except perhaps for the Far East, thisis the only record of traditional use of a microbi-al biomass as a food for human consumption.The aim of this review was to collate the infor-mation available in the case of the two speciesthat appear to have been utilized as a foodsource, S. platensis and S. maxima. It is hopedthat this information may encourage further in-vestigation on the different aspects of the lifeand the possible exploitation of these organisms.This work may, in turn, be of some help inalleviating one of the most pressing problemsnow facing mankind.

OCCURRENCE AND ISOLATIONSpirulina is a ubiquitous organism. After the

first isolation by Turpin in 1827 from a freshwa-ter stream (159), species of Spirulina have beenfound in a variety of environments: soil, sand,marshes, brackish water, seawater, and freshwa-ter. Species of Spirulina have been isolated, forinstance, from tropical waters to the North Sea(68), thermal springs (6), salt pans (52), warmwaters from power plants (49), fish ponds (122),etc. Thus, the organism appears to be capable ofadaptation to very different habitats and colo-nizes certain environments in which life for othermicroorganisms is, if not impossible, very diffi-cult. Typical is the population by alkalophylic S.platensis of certain alkaline lakes in Africa andby S. maxima of Lake Texcoco in Mexico. Insome of these lakes Spirulina grows as a quasi-monoculture. In the case of the African lakes inthe Chad region, Iltis has conducted an exten-sive survey of the phytoplankton of the alkalinelakes, permanent or temporary (75). These bod-ies of water have been classified into threegroups according to their salt content, mostlycarbonates and bicarbonates. The lakes with asalt concentration of <2.5 g/liter presented avery varied microbial population composed ofChlorophyceae, cyanobacteria, and diatoms. Inthe mesohaline lakes, characterized by salt con-centrations ranging from 2.5 to 30 g/liter, the

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SPIRULINA, THE EDIBLE MICROORGANISM 553

cyanobacterial population became predominant,although many species were present (Synecho-cystis, Oscillatoria, Spirulina, Anabaenopsis)(74). In the lakes containing salt concentrations>30 g/liter, the cyanobacterial population be-came practically monospecific and Spirulinawas the only organism present in significantquantities (69, 70). Indeed, S. platensis wasfound in waters containing from 85 to 270 g ofsalt per liter, but growth seemed to be optimal atsalt concentrations ranging from 20 to 70 g/liter,and it is possible that the population of S.platensis found at the highest salt concentra-tions, such as in temporary ponds just beforedrying, was that of the cyanobacterial biomassestablished when the concentration of salts wasmuch lower (71). A detailed investigation of twolakes, Rombou and Bodou, both characterizedby a very alkaline pH (10 to 10.3 for Rombouand 10.2 to 10.4 for Bodou) but different saltconcentrations (13 to 26 g/liter for the formerand 32 to 55 glliter for the latter), seemed toconfirm that salt concentration plays a directrole in the growth of S. platensis. In LakeRombou, cyanobacteria represent <50% of thephytoplankton population, whereas in Lake Bo-dou they account for at least 80% of the totalpopulation. In addition, in the former, S. platen-sis represented major but not the only phyto-plankton component with extensive quantitativeseasonal variations, whereas in the latter S.platensis was practically the only cyanobacte-rium present. Indeed, with the exception of themonths of November and December, when S.platensis accounted for 80% of the plankton, inall other months this species represented thetotality of the biomass in Lake Bodou. Thecorrelation existing between salt concentration

and abundance of Spiriulina was confirmed in alater study on a group of lakes characterized bya lower salt concentration (5 to 14 g/liter) (74). Inthe lakes of this group, characterized by thehighest salt concentration, a variety of Spiri-lina, S. platensis var. minor, was practically theonly cyanobacterium present, whereas in thosewith a lower salt concentration Spiriulina waspresent but represented only a fraction of themicrobial population. In addition, in the latterlakes wide fluctuations were observed in therelative abundance of S. platensis var. minorthat accounted for, according to the season, 70to 2% of the total biomass.An analogous situation appears to exist in the

alkaline lakes of the Rift Valley in East Africa.These lakes too are characterized by very highpH, reaching, in certain cases, values close topH 11, and very high salt concentrations, partic-ularly sodium carbonate originating from thesedimentary volcanic deposits. In some of theselakes, such as Nakuru, Elmenteita, and theCrater Lake, in which the pH ranges from 9.4 to11, S. platensis and S. platensis var. minor arethe predominant microorganisms present (79,157). ("Two or three other members of Myxo-phyceae and a few Diatoms occur in the threelakes under consideration, but they do not con-stitute a conspicuous feature of the plankton"[128].) In a more recent survey of the photosyn-thetic rates in some of the lakes of this area, itwas confirmed that in Lakes Nakuru (pH 10.5),Elmenteita (pH 9.4), Reshitani (pH 10.1), andBig Momela (pH 10.4), S. platensis, its varieties,and S. laxissima represent the most abundant, ifnot the only, constituents of the phytoplankton.In only one lake, Lake Nakuru, was anothercyanobacterium, Chroococcius miniutius, found

FIG. 2. Collection of S. maxima in Mexico. Anonymous Spanish map of the 16th century depicting theharvesting of S. maxima and, possibly, other algae from Lake Texcoco (reprinted, with permission fromEnciclopedia de Mexico [4]).

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554 CIFERRI

in considerable amounts. Yet, on a per-cellbasis, even in this lake S. platensis was 5- to 10-fold more abundant than C. minutus (102). Inanother lake of the same area, Lake Simbi, S.platensis was the only organism that could bedetected, reaching, in the first meters from thesurface, a concentration of 200,000 cells per ml(each coil being composed of ca. 10 cells), whichwas responsible for the exceptionally high ratesof photosynthesis (up to 13 g of 02 produced/m3per h) (101). Similarly, two crater lakes in Ethio-pia, Lakes Kilotes and Aranguadi, both charac-terized by a high salt content and an alkaline pH,support a dense population of Spirulina (150). InLake Kilotes (pH 9.6), S. platensis is the pre-dominant organism, although it is accompaniedby an unidentified species of Chroococcus. InLake Aranguadi, characterized by a more alka-line pH (10.3), S. platensis is the only microor-ganism present and its abundance is such thatwaters appear deep green (in Abyssinian, aran-guadi means green). The high concentration ofS. platensis was responsible for the extremelyhigh photosynthetic rates (1.2 to 2.4 g of 02produced/M2 per h).

It must be stressed that in many of these lakesdramatic changes in the population of Spirulinamay result from fluctuations in the alkalinity andthe salt concentration of the water. For instance,in the case of Lake Nakuru, the alkalinity (ex-pressed as milliequivalents of HC03- + C022-per liter) was 296 in 1929, 205 in 1931, 1,440 in1961, and 122 in 1969 (102). A more recentinvestigation on the lakes studied by Rich (128)and Jenkin (79) has shown that the density ofSpirulina not only undergoes seasonal variationsbut also may be reduced from the predominant,or sole, component of the phytoplankton to aminor component of the biomass. Thus, thecontribution of Spirulina to the primary produc-tion may become almost negligible whereas thatof other species (e.g., benthic diatoms) becomespredominant (157). These changes, at times fullyreversible, may be of considerable importancefor the animal communities associated withthese lakes (see below). Of course the lakeswhose physicochemical conditions do not varymay maintain a stable and abundant populationof Spirulina as must be the case for some of thelakes in the Chad area and Lake Texcoco inMexico. For these lakes it must be assumed thatSpirulina has been the primary component of thebiomass at least for centuries. In conclusion,some species of Spirulina, notably S. platensisand S. maxima, are capable of growing in waterswhose chemical composition makes life for othermicroorganisms very difficult if not impossible.In these environments, Spirulina grows as aquasi-monoculture (Table 1). This does notmean, of course, that other microorganisms do

not grow at all, as demonstrated, for instance,by the finding that all species of Spirulina so farisolated even from the most alkaline lakes arealways contaminated by bacteria. The bacterialflora associated with the cultures of Spirulina isvaried but with a preponderance of gram-nega-tive rods (105). It is not known whether anymutualistic relations exist between these bacteriaand the cyanobacterium, although in the labora-tory axenic cultures of S. platensis grow as well,and perhaps even better, than nonaxenic ones(2, 105). In addition, although no comparison ofthe requirements (medium composition includ-ing trace elements, alkalinity, light, etc.) ofaxenic cultures and nonaxenic ones has beenperformed, all data so far available indicate thataxenic cultures grow at least as well as nonax-enic cultures in the media and under the condi-tions developed for the latter isolates (2, 105,106, 126, 160). The bacteria associated with thecyanobacterial trichomes may be easily isolatedby plating trichomes on standard bacteriologicalmedia in which the pH has been adjusted to 9 to9.2. If media with pH close to neutrality areused, very few bacterial colonies are found. It ispossible to distinguish two main groups of bacte-rial contaminants, that of the organisms presentmostly in the culture medium and loosely adher-ing to the trichomes and those, called epiphyticcontaminants, bound or tightly adhering to thethin sheath enclosing the trichomes. Washingthe trichomes with aqueous solutions removesthe first group of bacteria without significantlyaffecting the second one. Fragments of tri-chomes containing no bacteria and hence ame-nable to the establishment of axenic culturesmay be obtained by extensively washing thetrichomes with sterile solutions followed by me-chanical fragmentation to give single cells orshort filaments containing two to four cells each.The fragments are then irradiated with UV lightand, after further washings, used to inoculatetest tubes of minimal medium in such a way as togive a concentration of ca. one cell per tube.After incubation in the light, a few tubes (3 to5%) contain viable cultures of S. platensis thatappear, even after repeated subculture, to bedevoid of detectable bacteria (105, 127). Recent-ly, a strain of S. platensis has been reported tohave been rendered axenic simply by repeatedstreakings on agar plates (21). However, thepossible presence of bacteria was assessed onstandard microbiological media, that is, presum-ably on media whose pH is not suitable forgrowth of the bacteria associated with the tri-chomes. The availability of axenic cultures al-lowed it to be established that, although S.platensis cannot grow under heterotrophic con-ditions, mixotrophic cultures are possible, con-sistently giving yields higher than those of cul-

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VOL. 47, 1983 SPIRULINA, THE EDIBLE MICROORGANISM 555

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tures grown under photoautotrophic conditions(see below).Due to the alkalinity of the growth medium,

the microbial load of S. platensis cultures hasbeen reported to be one order of magnitudelower than that of eucaryotic algae, such asScenedesmus acutus, which grow in acid media(10). However, a quantitative study of the bacte-rial flora associated with open-pond cultures ofS. platensis and S. maxima has shown that thecontaminating bacteria may account for ca. 1%of the total biomass (100). If the trichomes arewashed repeatedly with sterile physiological so-lution, the bacterial contribution to the totalbiomass becomes negligible (<103 bacterial cellsper trichome) (127). Thus, harvesting of culturesby filtration or centrifugation followed by wash-ing may result in biomasses that contain insig-nificant amounts of bacterial contaminants. Mi-crobiological investigations of samples of Dihdobtained from local markets in the Chad area haveindicated the presence of aerobic and anaerobicbacteria, and fecal streptococci have been isolat-ed from S. maxima harvested from Lake Texco-co (76). As expected, the number of all viablebacteria, including those that may represent adanger to human health, decreases in the driedsamples even in those processed by sun drying(10, 76).

MORPHOLOGY AND TAXONOMYSpirulina is a multicellular, filamentous cy-

anobacterium. Under the microscope, Spirulinaappears as blue-green filaments composed ofcylindrical cells arranged in unbranched, helicoi-dal trichomes (Fig. 3). The filaments are motile,gliding along their axis. Heterocysts are absent.The helical shape of the trichome is character-

istic of the genus but the helical parameters (i.e.,pitch length and helix dimensions) vary with thespecies, and even within the same species, dif-ferences have been observed in these parame-ters (98, 128) or may be induced by changing theenvironmental conditions such as growth tem-perature (162). The helical shape is maintainedonly in liquid media, and in solid media thefilaments become true spirals (164). The transi-tion from a helix to a flat spiral is a slow processdepending on the water content of the agarsurface, whereas the reverse occurs almost in-stantly when, for instance, a drop of water isdeposited on the agar surface in contact with aspiral. The transition from helix to spiral isprobably related to the necessity of reducing, insolid media, the surface area exposed to air. It ispossible that these transitions are caused byhydration or dehydration of the oligopeptides inthe peptidoglycan layer, resulting in changes inthe rigidity of the cells (165).The diameter of the cells ranges from 1 to 3

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p.m in the smaller species and from 3 to 12 p.m inthe larger ones. Comparison of an authenticisolate of S. platensis from Chad and of S.maxima from Mexico, grown in the laboratoryunder identical conditions, showed that S. maxi-ma is characterized by a diameter of the helix of50 to 60 p.m and pitch of 80 pm; values >35 to 50p.m and 60 pm, respectively, were observed forS. platensis. On the other hand, cell dimensionswere greater in S. platensis than in S. maxima(diameter, 6 to 8 p.m in the former and 4 to 6 p.min the latter) (97). The cytoplasm of the smallerspecies appears homogeneous, with no gas vac-uoles or inclusions and scarcely visible septa.On the contrary, the larger species such as S.platensis and S. maxima have a granular cyto-plasm containing gas vacuoles and easily visiblesepta. As will be discussed later, the presence orabsence of the septa has been one of the distin-guishing characters used in the classification andgeneric assignment of these organisms. Tri-chomes are, in general, a few millimeters long,although under certain conditions trichomes ofS. platensis as long as 20 mm have been ob-served (162).Among the macromorphological variations

occurring in Spirulina, the appearance of iso-lates with straight trichomes has been reportedto occur spontaneously or after mutagenesis.This has induced Bourrelly (16) to considerSpirulina an Oscillatoria, deserving, at most, thestatus of a subgenus within the family Oscillator-iaceae. However, as discussed below, this opin-ion is not widely accepted.

Electron microscopy of ultrathin sections ofS. platensis revealed that the cell wall is com-posed of possibly four layers (98, 160). The mostexternal or outer membrane layer (L-IV) iscomposed of material arranged linearly in paral-lel with the trichome axis and is consideredanalogous to that present in the cell wall ofgram-negative bacteria. Layer III is possiblycomposed of protein fibrils wound helicallyaround the trichomes, whereas the peptidogly-can-containing layer (L-II) folds towards theinside of the filament, giving rise, together with aputative fibrillar inner L-I, to the septum sepa-rating the cells. However, if layers I and III wereartifacts arising during the preparation of thesamples for electron microscopy (38), the sep-tum separating the cells would be composed ofthe peptidoglycan layer only. The septum ap-pears as a thin disk, folded in part. This foldcovers a portion of the septum surface, and itsextent seems to be related to the pitch of thetrichome; the larger the pitch, the smaller thefolded area and vice versa. Indeed, whereas inS. platensis the fold covers ca. 5% of the totalseptum area, in S. laxissima, characterized by amuch larger pitch, the fold covers ca. 3% of the

MICROBIOL. REV.


septum area. Stretching of the filament results inthe disappearance of the septal fold that, indeed,is apparently absent in nonhelical cyanobacteria(165).The most prominent cytoplasmic structure

is the system of thylakoids originating fromthe plasmalemma (57, 64, 97, 162) but quitedistinct from the well-evident mesosomes (3,154). At times, the thylakoids appear to bearranged in concentric whorls especially evidentin adult cells. During cell division, the invagina-tion of the plasmalemma, from the outer portiontowards the cell center, is accompanied bybreakage of the thylakoids that thus becomedistributed between the two daughter cells. Phy-cobilisomes, high-molecular-weight aggregatesof phycocyanins, appear to be attached to thethylakoids (162), as expected on the basis oftheir function as light-harvesting antennae.Cyanophycin granules, a reserve material

composed in other cyanobacteria of copolymersof amino acids in general chains of poly-L-aspartic acid with arginine attached to the ,B-carboxyl groups, are present in S. platensis,their relative amount varying with the mediumcomposition, cell age (163), and growth tempera-ture (162). Polyglucan granules, cylindrical bod-ies, carboxysomes, and mesosomes have alsobeen detected (3, 98, 154, 162, 163). Van Eyke-lenburg described these organelles in S. platen-sis and evaluated their occurrence in culturesgrown at different temperatures and light condi-tions (162) and in media containing differentconcentrations of nitrate (163). At low tempera-tures, when the demand for amino acids islimited by the reduced growth rate, cyanophycingranules are the most abundant organelles, oc-cupying up to 18% of the cell volume. Onincreasing the growth temperature, the contentin these granules progressively decreases, and at25 to 30°C they are practically undetectable.Polyglucan granules were most prominent at lowtemperatures (15 to 17°C), decreasing in concen-tration at higher temperatures. The relative con-centration of the other cell organelles was notinfluenced significantly by light or temperature.Carboxysomes, polyhedral bodies containing

ribulose-1 ,5-bisphosphate carboxylase, werepresent only when S. platensis was grown athigh light intensities and in media containing ahigh nitrate concentration. At low light intensi-ties or at low nitrate concentrations, carboxy-somes disappeared, thus supporting the viewthat these organelles may represent some sort ofstorage bodies for ribulose-1,5-bisphosphatecarboxylase and, possibly, other proteins. Gasvesicles, in the shape of hollow cylinders withcone-shaped ends, with a diameter of ca. 65 nmand a length of up to 1 ,um were easily detected.The vesicle membrane consists of coils of pro-

tein molecules, possibly all of the same type,arranged in ribs spaced 4 to 5 nm apart. Theorganelles are responsible for cell buoyancy andhence for distribution of the organism along thewater column (174a). Unfortunately, no infor-mation is available on the relative abundance ofthese organelles in Spirulina or on their variationin relation to changes in the environmental con-ditions, especially light intensity and dissolvedoxygen content.A number of "unusual inclusions" have been

reported in a cytological study of 60 differentcyanobacteria, representing at least 30 speciesand including two isolates of Spirulina (82). Thesignificance of these inclusions could not beassessed since many were present in a few of thecyanobacteria examined and, at times, wereunique to one or a few species. The authorssuggested that some, perhaps many, of these"inclusions" were due to infection by intracellu-lar symbionts or viruses. This may be the casealso of rhapidosomes, organelles of uncertainorigin and function, perhaps components of aputative motility organelle or parts of an incom-plete phage, which have been observed in anunidentified species of Spirulina (24) but not inS. platensis (162). Since rhapidosomes havebeen characterized in the flexibacterium Sapro-spira grandis (32), the occurrence in Spirulinawould have strengthened the similarities be-tween Spirulina and Saprospira, the latter beingconsidered by some authors as an apochlorotic,nonphotosynthetic cyanobacterium (92).The life cycle of Spirulina in laboratory cul-

ture is rather simple (Fig. 4). A mature trichomeis broken in several pieces through the formationof specialized cells, necridia, that undergo lysis,giving rise to biconcave separation disks. Thefragmentation of the trichome at the necridiaproduces gliding, short (two to four cells) chainsof cells, the hormogonia, that move away fromthe parental filament to give rise to a newtrichome. The cells in the hormogonium lose theattached portions of the necridial cells, becom-ing rounded at the distal ends with little or nothickening of the walls. During this process, thecytoplasm appears less granulated and the cellsassume a pale blue-green color. The number ofcells in hormogonia increases by cell fissionwhile the cytoplasm becomes granulated, andthe cells assume a brilliant blue-green color. Bythis process trichomes increase in length andassume the typical helicoidal shape. Random butrare spontaneous breakage of trichomes togeth-er with the formation of necridia assure growthand dispersal of the organism. Akinetes have notbeen reported.

Determination of the taxonomic position ofSpirulina has proved to be rather difficult. Thegenus Spirulina was established in 1827 by Tur-

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FIG. 3. Morphology of Spirulina. (A) Optical microscopy (X400) of axenic S. platensis. (Photo by G.Caretta.) (B) Scanning electron micrograph of a trichome of axenic S. platensis. (Photo by R. Loc(i.) (C)Scanning electron micrograph of a portion of a trichome of axenic S. platensis. (Plhoto by R. Locci.) (D) Scanningelectron micrograph of nonaxenic trichomes of S. inaxima. (Plhoto by R. Locci.)

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562 CIFERRI

FIG. 4. Life cycle of Spirulina. For details, see

text. (Redrawn from reference 8.)

pin for S. oscillarioides (159). Since Turpin didnot mention the presence of septa in his isolate,in 1852 Stizenberger created the genus Arthro-spira for the spiral cyanobacteria in which septawere clearly visible (149). This distinction,which also separates the larger forms from thesmaller ones, was maintained for another 100years. Thus, for instance, Gomont in 1892 (53)grouped in the genus Arthrospira Stizenbergerthe forms with visible septa and larger dimen-sions, such as S. platensis, whereas he reservedthe genus Spirulina Turpin for the smaller formsin which septa were not visible and thus were

characterized by "unicellular trichomes." In1917, Gardner (49) questioned the validity of thepresence of septa to distinguish between Arthro-spira and Spirulina since he had "determinedrepeatedly that this distinction can no longermaintain." However, "for the sake of conve-

nience," he suggested retaining the name Arth-rospira for the forms "with conspicuous cross-walls" and that of Spirulina for those "withobscure cross-walls." Although a few yearslater Figini showed that, with appropriate stains,septa could be demonstrated in fresh and dryspecimens of 13 different species of Spirulina,including the most minute ones such as S. subti-lissima (47), Crow reported that, indeed, incertain Spirulina septa could be demonstratedbut that it was impossible to do so in otherisolates (30). The former, even if they had thetypical small dimensions of a Spirulina, had tobe grouped with Arthrospira, and the name

Spirulina was to be reserved for the aseptateforms. This position was also that of Geitler (50),who revised the classification by grouping the

two genera in a single one, Spirulina, subdivedin section I, Arthrospira, for the "large formswith transverse wall which are seen in livingalgae" and section II, Euspirulina, for the small-er forms with invisible septa. The separation ofSpirulina and Arthrospira was accepted as validup to 1959 (35). Finally, in 1961 Welsh (176)clearly pointed out that "whether or not thetransverse walls can be seen depends on wheth-er the alga is dead or alive and on the microscop-ical technique used. With today's phase-contrast[microscopy] much can be seen which at thetime of publication of Geitler's work was invisi-ble." Thus he suggested that the generic nameSpirulina, older than Arthrospira, be usedregardless of the presence of visible septa thatcan, of course, be easily detected even in Spiru-lina by electron microscopy (64). Yet, as late as1968, Drouet (39) still maintained the two ge-nera, Arthrospira for the forms with apparentcross walls and Spirulina "for the forms appear-ing unicellular without easily demonstrablecross walls." In addition, much to the confusionof the nonspecialist, due to differences in themorphology of the outer wall of the terminalcells, both S. maxima and S. platensis areplaced in the genus Microcoleus, as M. lyngbya-ceus.To make things worse, 2 years later, Bourrelly

(16) concluded that "only the character of thehelicoidal shape of the trichome separates Spiru-lina from Oscillatoria." Since certain Oscilla-toria have portions of the trichome that appearhelicoidal and the degree of spiralization ofcertain Spirulina may vary (72, 128) and, attimes, even straight trichomes are evident (93,98), Bourrelly suggested considering Spirulina asubgenus of Oscillatoria. Thus, S. platensisshould be named 0. platensis and S. geitleri(synonymous with S. maxima) should be named0. pseudoplatensis (the names of 0. maximaand 0. geitleri being used to designate othercyanobacteria) (16, 72). However, more recentlyRippka and co-workers (134) emphasized thatthe helical shape of Spirulina "is a stable andconstant property" of the genus that permits itsdifferentiation from the other groups of filamen-tous, non-heterocystous cyanobacteria: Oscilla-toria, Pseudanabaena, and the LPP (Lyngbia,Phormidium, and Plectonema) group (135). Inaddition, differences between Spirulina and Os-cillatoria have been reported in the genome size(59), chemical composition (especially in thecase of fatty acids [see later]), antigenicity (22),and ultrastructure (154). Thus, it does not seemunreasonable to maintain the genus Spirulina forat least the cyanobacteria characterized by high-ly spiralized trichomes living in freshwaters withhigh salt concentrations and alkaline pH; that isthe object of this review. In addition, the names

MICROBIOL. REV.


S. platensis and S. maxima (= S. geitleri) arethose utilized in practically all the recent litera-ture dealing with these two cyanobacteria.

PHYSIOLOGYLike most cyanobacteria, Spirulina is an obli-

gate photoautotroph and cannot grow in the darkin media containing organic sources of carbon(85, 105). However, in the light it may utilizecarbohydrates since, for instance, the additionof 0.1% glucose to the growth medium enhancesgrowth rate and cell yield (106). Especially indim light, mixotrophic growth results in cellyields that are two- to threefold higher than thecorresponding yields obtained photoautotrophi-cally. Peptone at the same concentration wasless effective but glucose and peptone addedtogether seemed to exert a synergistic effect(105). The utilization of glucose was verified bysupplying cultures of S. platensis with [14C]-glucose (106). Within less than 4 days of culture,all labeled glucose disappeared from the mediumand almost 50% of the label was recovered withthe cells, the rest being released either as CO-(34%) or as organic by-products excreted intothe medium (19%). In these experiments aninteresting phenomenon, the so-called mixotro-phic lysis, was observed. If the size of theinoculum was kept small (i.e., cultures inoculat-ed to give an initial optical density at 560 nm of<0.1 at 1-cm light path), after a brief period ofgrowth, the cultures stopped growing and thecells underwent complete lysis. On the otherhand, growth was normal if the culture wasinoculated to an initial optical density of ca. 0.2.No explanation for the phenomenon of mixotro-phic lysis has been offered but it has beenobserved that, before lysis, cells from a culturestarted with a "small" inoculum contained six toseven times more alkaline protease activity thancells from cultures with a "large" inoculum orcells from photoautotrophic cultures. Mixotro-phic lysis could be prevented by depriving themedium of Mn ions. Both growth rate and cellyield may be increased under photoautotrophicconditions by increasing the amount of nitrogensupplied (up to 120 mM nitrate), especially whencultures were performed at temperatures (35 to37°C) higher than the optimal ones (32 to 35°C)(163). In the laboratory, cultures of S. platensisshow a wide pH optimum (8 to 11), but growth isevident also at pH values close to 7 and as highas 11.3 (54). The optimal light intensity wasfound to be between 20 and 30 klx (105). Underfield conditions, optimal day temperature hasbeen reported to be around 40°C and the nighttemperature is around 25°C (see below). Above40°C the cultures do not grow. Laboratory cul-tures kept at 45°C for up to 24 h do not grow, butgrowth is resumed when the culture is brought

back to 350C. Above 450C, massive breakage ofthe trichomes followed by cell lysis has beenobserved. Even a brief period (e.g., 10 min) ofexposure at temperatures around 50°C results indeath of the cultures (25). Compared with cellsgrown at suboptimal light concentrations, cellsgrown at ca. 20 klx have a higher content ofcarotenoids. A slight increase was also observedin the case of chlorophyll, whereas no differencewas noticed in the case of phycocyanins (106).With the view of obtaining massive cultures of

Spirulina utilizing seawater, attempts have beenmade to cultivate S. platensis and S. mnaxima inmedia containing seawater in part or in toto.Although analogies exist between the salt com-position of seawater and that of the alkalinelakes of Africa and Lake Texcoco (8), the lowcontent in carbonates, phosphates, and com-bined nitrogen together with the high concentra-tions ofMg and Ca make seawater unsuitable formass culture of Spirulina. Not only is growth ofSpirulina inhibited by high concentrations of Mg(177), but also the addition of carbonates orphosphates or both causes precipitation of theirCa and Mg salts. A preliminary removal of thesetwo cations allows addition of carbonates, phos-phates, and nitrates (43). However, probably thepretreatment to remove Mg and Ca ions wouldrender the process economically unacceptable.

BIOCHEMISTRYThe phycocyanins, biliproteins involved in the

light-harvesting reactions, have been resolvedby gel electrophoresis in S. platensis and S.maxima (29) and isolated from the former (17).Both c-phycocyanin and allophycocyanin ap-pear to be oligomeric complexes composed of atleast two different subunits that may be resolvedby electrophoresis under denaturing conditions.The a- and 1-subunits of c-phycocyaninsshowed mobilities corresponding to molecularweights of 20,500 and 23,500, respectively, re-sulting in an oligomer with a minimum molecularweight of ca. 44,000. Allophycocyanin wasfound to be composed of subunits with molecu-lar weights of ca. 18,000 and 20,000 to give anoligomer with a minimum molecular weight ofca. 38,000. Absorption and fluorescence spectrawere similar to those reported for c-phycocya-nins and allophycocyanins isolated from othercyanobacteria. A study of the denaturation andrenaturation of c-phycocyanin indicated the pos-sibility that more than one chromophore existsin this biliprotein (143).Phycocyanins may serve also as a storage

material since it has been found that the phyco-cyanin concentration was highest when S. pla-tensis was cultivated under favorable nitrogenconcentrations (18). If the level of availablenitrogen in the medium decreased, or the cul-

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564 CIFERRI

tures were completely deprived of nitrogen, acorresponding decrease in the phycocyanin con-tent was observed. No other nitrogen-containingcompounds decreased under these conditions,and the decrease of phycocyanin concentrationwas associated with an increase in the activity ofa protease acting on purified c-phycocyanin. If,under these conditions as well as after inhibitionof protein synthesis, the cellular concentrationof phycocyanins decreased, severe inhibition ofphotosynthesis and growth was observed. Ribu-lose-1,5-bisphosphate carboxylase, accountingfor ca. 12% of the soluble protein of S. platensisand S. maxima, was purified and partially char-acterized (138). By gel electrophoresis the mo-lecular weight of the enzyme was estimated tobe ca. 500,000, a value similar to that reportedfor the enzyme isolated from chloroplasts ofhigher plants and unicellular algae as well assome cyanobacteria (148). Electrophoresis un-der denaturing conditions of the enzyme from S.maxima revealed the presence of two types ofsubunits, a larger one with an apparent molecu-lar weight of ca. 55,000 and a smaller one of ca.12,000. Thus, it is quite likely that the holoen-zyme is composed of eight large subunits ar-ranged around a "core" of eight small subunits.The amino acid composition of ribulose-1,5-bisphosphate carboxylase purified from S. maxi-ma was found to be very similar to that reportedfor the enzyme isolated from higher plants andunicellular algae. Thus, just like the enzymefrom higher plants such as tobacco (41), ribu-lose-1,5-bisphosphate carboxylase from Spiru-lina also may be considered as a promisingsource of high-quality protein for human nutri-tion.Cytochrome C554, a cytochrome with high

redox potential that links photosystems I and II,has been purified from S. platensis (178) and S.maxima (63). The molecular weight of the pro-tein was found to be ca. 10,000, like that ofcytochrome C554 from other cyanobacteria, uni-cellular algae, and higher plants. Another cyto-chrome involved in photosynthesis, cytochromef, was purified from S. maxima (5, 62, 63) and S.platensis (139). The molecular weight was ca.38,000, a value close to that of cytochrome fisolated from spinach chloroplasts. Similarly,striking similarities were observed in the aminoacid composition (62).

Ferredoxin, one of the electron carriers ofphotosynthesis, was purified from S. maxima(55) and sequenced from S. maxima (151) and S.platensis (174). As the protein isolated fromhigher plants, S. maxima ferredoxin containedtwo atoms of Fe and two atoms of sulfur permole. Optical adsorption, other spectral charac-teristics, and oxygen evolution assays confirmedthe close similarity existing between the ferre-

doxin isolated from the cyanobacterium and thatisolated from higher plants or algal chloroplasts.The similarity between the ferredoxin from Spir-ulina and that from chloroplasts was substantiat-ed by immunological tests (152). The stability,especially at room temperature, of the ferredox-in isolated from S. maxima was much higherthan that of the protein isolated from higherplants and unicellular algae. This characteristic,together with the ease of isolation from driedcells and even spray-dried commercial prepara-tions (55), render Spirulina a promising sourceof ferredoxin. Ferredoxin II, another ferredoxincharacterized by a different redox potential andpresent in smaller amounts, was isolated in asubsequent investigation (20, 63). The presenceof two different ferredoxins may be ascribed tothe other biochemical activities associated withthese electron carriers (donor of electrons tonitrite reductase, sulfite reductase, glutamatesynthase, electron acceptor in the phosphoclas-tic cleavage of pyruvate, etc.) for which eachtype of ferredoxin may be more suited. Theamino acid sequences of the last 23 amino acidsfrom the amino-terminal position of the ferre-doxins (not specified, but probably type I) fromS. platensis and S. maxima appear to be almostidentical, differing in only one amino acid (144).A coupling factor complex, linking phosphoryla-tion to electron transport, was also partiallypurified from S. platensis (109).A preliminary characterization of a cyanide-

insensitive superoxide dismutase from S. platen-sis indicated the presence of Fe, as in some ofthe bacterial and cyanobacterial dismutases,rather than Cu or Zn as found in the enzymesfrom chloroplasts (94, 95).The only data on the transport of inorganic

nutrients in Spirulina concern sulfur. S. platen-sis appears to possess an active, energy-depen-dent transport system for sulfate. Under photo-autotrophic conditions, probably two sulfatepermeases are present. One permease is consti-tutive, as found for the only other cyanobacter-ium studied, Anacystis nidulans, whereas theother is inducible like that of heterotrophic bac-teria, fungi, and some higher plants (103).A number of mutants of S. platensis resistant

to two analogs of phenylalanine (P-thienylalan-ine and p-fluorophenylalanine), methionine(ethionine), proline (azetidin-2-carboxylic acid),and tryptophan (5-fluorotryptophan) were re-cently isolated and partially characterized (126).A few mutants appear to be resistant to oneanalog only, whereas the majority seem to havebecome resistant simultaneously to the analogsof phenylalanine, methionine, and proline butnot of tryptophan. All of the cross-resistantmutants analyzed appear to overproduce therespective parental amino acids and thus be-

MICROBIOL. REV.


come resistant to the analogs by reducing theiruptake into the cells and their incorporation intoprotein (124). It appears likely that in thesestrains a mutation in one enzyme at the begin-ning of a metabolic branch point results in analteration in the mechanisms regulating its activ-ity. This may lead to overproduction of groupsof amino acids. Such a pattern of metaboliccontrol, the so-called endo-oriented control be-cause of being most sensitive to endogenousproducts, is typical of cyanobacteria in contrastto the "exo-oriented" control (most sensitive toexogenous products) that is typical of manyheterotrophic bacteria (82). Indeed, obligatephotoautotrophs, like most cyanobacteria, arebarely capable of utilizing exogenous aminoacids, for which some of them appear even tolack an active transport system (56). The major-ity of the mutants of S. platensis so far isolatedare presumably deregulated at a proximal meta-bolic branch point rather than at the terminalbranchlet, resulting in the production of morethan one end product and, as a consequence,resistance to analogs of different amino acids.However, a mutant resistant to azetidin-2-car-boxylic acid was found to be resistant to thisanalog only and to overproduce only proline(124). A mutant resistant to ethionine did notoverproduce any amino acid and had an alteredmethionyl-tRNA synthetase (125). The mutant'senzyme, unlike that from the parental strain, hada reduced affinity for the analog so that itsincorporation into protein, in place of methio-nine, was practically reduced to nil. Finally, theonly mutant resistant to 5-fluorotryptophanseemed to possess an altered tryptophanyl-tRNA synthetase. Thus, in S. platensis, muta-tions conferring resistance to amino acid analogsmay involve the regulation of amino acid biosyn-thesis or mechanisms for the uptake and incor-poration of amino acids into protein. Although ina wild-type strain the cellular pool of free aminoacids has been reported to be very small (177),many of the overproducing mutants excrete dur-ing growth only a portion of the amino acid(s)overproduced and >50% is released in the medi-um at cell lysis (126). This finding renders attrac-tive the utilization of some of these mutants formass cultures. Since, at least under laboratoryconditions, most of the mutants grow at thesame rate and attain the same cell concentrationas the wild-type strain, it is conceivable toproduce S. platensis cells with different qualita-tive-quantitative levels of selected amino acidsand, possibly, other metabolites. It would bepossible, for instance, to improve the nutritionalvalue of S. platensis by compensating for thelow content in methionine (see below) throughthe use of mutants that have higher intracellularpools of this amino acid. Since in higher plants

and unicellular organisms overproduction ofproline is one of the mechanisms responsible forstress resistance, including the presence of highsalt concentrations, the mutants of S. platensisoverproducing proline were analyzed for thecapacity to grow in media containing high con-centrations of sodium chloride (123). All mu-tants overproducing proline grew in media con-taining NaCl concentrations that inhibitedgrowth of the parental strain. In addition, apositive correlation was found between theamount of proline overproduced and the degreeof osmotolerance, suggesting the possibility thatthese mutants may be utilized for cultures inbrackish waters unsuitable for the strains of S.platensis so far utilized.

Little is known concerning the mechanismsfor genetic recombination in cyanobacteria, es-pecially in the case of the filamentous species.Indeed, whereas there are a few reports demon-strating the occurrence of transformation in theunicellular forms (90), for the filamentous cya-nobacteria there is only one report giving indi-rect evidence for the presence of transformationin Nostoc muscorum (155). As far as I am aware,no information is available for Spirulina or forany of the Oscillatoriaceae, yet it is likely that amechanism for genetic recombination may existalso in these cyanobacteria. Spheroplasts, whichmay now be prepared efficiently in S. platensis(136), may be of considerable help in attemptingheterologous fusions, a technique successfullyused for other procaryotes, such as streptomy-cetes, recalcitrant to conventional genetic re-combination.So far no phage infecting Spirulina or other

Oscillatoriaceae has been isolated (110, 137) norhave plasmids been found in S. maxima and S.platensis (G. Riccardi, unpublished data). How-ever, one may hope that phages or plasmids maybe found eventually or that plasmids with selec-table markers now constructed for other cyano-bacteria (e.g., A. nidulans [89]) and also capableof transforming bacteria may be utilized in thecase of Spirulina.The genome sizes of 128 strains representing

all major taxonomic groups of cyanobacteriahave been determined by renaturation kineticsanalysis (59). The genome sizes could begrouped into four classes that could correspondto a progressive duplication of an "ancestralgenome' of ca. 1.2 x 109 daltons to give ge-nomes two, three, four, and six times this ances-tral genome. The value of 2.53 x 109 daltonsfound for an unidentified species of Spirulina,and close to the values reported for many bacte-ria such as Escherichia coli would correspond totwo copies of the putative ancestral genome.The DNA base compositions of the only twoisolates of Spirulina analyzed have been report-

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566 CIFERRI

ed to be 44 and 52 mol% guanosine plus cytosine(60). The lower value is close to that found for 10isolates of Oscillatoria, the higher one clearlydifferentiating this isolate from the other Spiru-lina as well as from Oscillatoria. However, forthe moment at least, Rippka et al. (134) considerboth isolates as Spirulina even if they showgross morphological differences, one being char-acterized by thick filaments with many gas vacu-

oles and the other having much thinner filamentsand no observed vacuoles. In addition, the habi-tats were also different, one being a marineisolate and the other growing in brackish waterbut not in seawater.

CHEMICAL COMPOSITIONAlready the first analyses performed on Dihe

indicated a high protein content: 45% of the dryweight in the samples analyzed by Leonard andCompere (91) and 62% in laboratory-grown S.platensis (28). More recent analysis confirmedthat protein represents more than 60% and, incertain samples, even 70% of the dry weight(Table 2). The protein content of Spiriulina ap-

pears to be high also when compared with that ofunicellular algae and other cyanobacteria. For10 species of eucaryotic algae, protein account-ed for 10 to 46% of the dry weight, whereas fourcyanobacteria gave values between 42 and 51%(19). However, values close to 60% have oftenbeen reported for the strains of Chlorella andScenedesmus that have been extensively studiedas possible sources of alimentary protein (1,175).An exhaustive study was performed by Pao-

letti et al. (115) of the chemical composition of S.platensis and S. maxima grown in the laboratoryor in open ponds (Table 3). For both species,cells grown in the laboratory contained more

protein than cells grown in open ponds; thelatter, on the other hand, contained a higherpercentage of carbohydrates and ash. In thesame investigation, a comparison was made of

TABLE 2. Approximate composition of S.platensis, S. maxima, and soybean meal

% of dry wt

Sample Crude Crude Crude CrudeWater Ash liisfbrcarbohy-prtilipids fiber drates prote

S. platensis' 6-10 4-5 9-14 3-8 10-18 56-77S. maximab 4-7 6-9 4 1 8-13 60-71Soybeanmeal 7-10 4 16-20 3-5 19-35 34-40

" Laboratory and pond grown, lyophilized or drumdried. Data from reference 115.

b From Lake Texcoco, spray dried. Data from refer-ence 40.

TABLE 3. Approximate composition of S. platensisand S. maxima grown in the laboratory and in open

ponds% of dry wt"

Component S. platensis S. naximanLaboratory Pond Laboratory Pond

Crude protein 64-74 61 68-77 60Crude lipids 9-14 12 9-14 15Crude 12-20 19 10-16 16

carbohydratesAsh 4-6 8 4-6 9

" Data from reference 115.

the chemical composition of both species growneither in open ponds or in polyethylene tubes(see below). Cultures from open ponds con-tained more protein and less carbohydrate thanthose grown in the closed system.The amino acid spectrum of Spirulina protein

is similar to that of other microorganisms (83,175) and, in comparison to standard alimentaryproteins such as those of eggs or milk, it issomewhat deficient in methionine, cysteine, andlysine (1, 10, 28). The amino acid compositionsof S. platensis and S. maxima grown in openponds or in polyethylene tubes have been evalu-ated (46, 115). Compared with the protein stan-dard elaborated by the Food and AgricultureOrganization or egg albumin, variations werereported in the content of a number of aminoacids, with significant differences in the case ofthe above-reported amino acids. However, thevarious authors conclude that, as a proteinsource, Spirulina, albeit inferior to standardalimentary protein such as meat or milk, issuperior to all plant protein including that fromlegumes. Thus, it appears that the high concen-tration of protein together with its amino acidcomposition make Spirulina a source of noncon-ventional protein of considerable interest.Because uric acid is produced in humans and

other mammals in the metabolism of purines andhigh levels of this metabolite may cause patho-logical conditions such as gout, a constant worryin the utilization of microbial cells as food orfeed has been their high nucleic acid content. InS. maxima and S. platensis, RNA has beenreported to represent 2.2 to 3.5% of the dryweight, whereas DNA represents 0.6 to 1% (10,27, 140). The total nucleic acid content, there-fore, is <5% of the dry weight, a value close tothat reported for unicellular algae, such as Chlo-rella and Scenedesmus (4 to 6%), but definitelylower than that of bacteria or yeasts (4 to 10%but up to 9 to 22% when the microorganisms arecultivated at the high growth rates required forindustrial production) (99). Thus, whereas a

MICROBIOL. REV.


typical single-cell protein (SCP), such as yeastgrown on alkanes, contains at least 1 g of nucleicacids per every 10 g of protein (65), S. maximaand S. platensis grown in the laboratory or inopen ponds contain 0.6 to 0.7 g per 10 g ofprotein (115).

Considerable variations have been reported inthe fatty acid content. Although differences havebeen demonstrated in the lipid content of S.platensis and S. maxima or in that of laboratory-and pond-grown cultures (115), the wide varia-tions reported in the literature (1.5 to 12% of thedry weight) (44, 66, 113, 118, 140) indicate thatthere may have been striking differences in theprocedures for the extraction or the estimation,or both, of the lipid content. In S. platensis andS. maxima free fatty acids account for 70 to 80%of the total lipids, the remaining being chieflymono- and digalactosyl glycerides and phospha-tidyl glycerol (66). Interesting variations havebeen observed in the degree of unsaturation ofoctadecatrienoic acid (86), and attempts havebeen made to utilize the presence of the differentisomers of this acid as a taxonomic criterion.The presence of high concentrations of -y-lino-lenic acid, synthesized in S. platensis by directdesaturation of linoleic acid, seemed to be char-acteristic of Spirulina, the a-isomer being pre-dominant in the other cyanobacteria and leaflipids (104). The high content of these twoessential fatty acids of possible considerableimportance for human nutrition (components ofthe so-called vitamin F), even in commercialpreparations of S. maxima (66), may be of someinterest for the utilization of Spirulina as a food.More recently, -y-linolenic acid was reported tobe present in other cyanobacteria, e.g., instrains of unicellular Synechococcus, Aphano-capsa, and Microcystis (84) as well as in filamen-tous ones such as Oscillatoria (85). Further, thelatter investigators reported that, of the threeisolates of Spirulina examined, one had a pre-dominance of at-linolenic acid. Therefore, for themoment at least, the presence of -y-linolenic acidin all isolates of Spirulina is in doubt, thusrendering its presence of uncertain importancefor taxonomic purposes. However, the fairlyhigh concentration of these two fatty acids in S.platensis and S. maxima remains of consider-able nutritional interest. Among the hydrocar-bons, n-heptadecane is the most abundant (65 to70%) in S. platensis and in S. maxima (44, 51,158), as in the majority of the other cyanobac-teria so far examined (51). Cholesterol and ,B-sitosterol are the main sterols present in S.maxima and S. platensis (96, 114, 140). Thepresence of these and other sterols was reportedto be somewhat related to an antimicrobial activ-ity of S. maxima (96) which, however, has neverbeen characterized (76).

Poly-,B-hydroxybutyrate, a reserve of carbonand energy in many bacteria, has been isolatedfrom S. platensis (21). The compound accumu-lates during exponential growth, reaching, at thebeginning of the stationary phase, a concentra-tion corresponding to 6% of the dry weight.Among the pigments, the most abundant is

chlorophyll a, the only chlorophyll present,which accounts for 0.8 to 1.5% of the dry weightin S. maxima and S. platensis (115). Mixoxanth-ophyll and ,-carotene are the major carotenoids(61, 111, 112), their content representing approx-imately 0.2 to 0.4% of the dry weight (111, 112,115, 140). The fairly high content of some ofthese pigments, possibly responsible for thecolor of the feathers of certain species of flamin-gos (see below), has stimulated the use of Spiri-lina also as a source of pigments for fish, chick-ens, and eggs.

Carbohydrates, accounting for 15 to 20% ofthe dry weight (23, 140), are represented in S.platensis essentially by a branched polysaccha-ride, composed of only glucose and structurallysimilar to glycogen (23). Another glucose-con-taining polysaccharide, representing ca. 1% ofthe dry weight, was also isolated and character-ized (161), whereas the presence of a rhamnan,reported to be the main polysaccharide of S.platensis (120), has not been confirmed (23).Finally, all vitamins have been found in S.platensis and S. maxima and their concentra-tions have been evaluated (27, 115, 140). Cyano-cobalamin appears to be rather abundant, reach-ing a concentration of up to 11 mg per kg of driedcells (27, 140).

PRODUCTIONThe only plant for large-scale production of

Spirulina operating at the moment is that ofLake Texcoco, in the Valley of Mexico. Thelake is located 2,200 m above sea level but in asemitropical climate (average yearly tempera-ture, 18°C). As already mentioned, S. maximagrows naturally in the lake, and the firm thatoperates a plant to extract soda from the lake isnow recovering and commercializing the cyano-bacterial biomass. S. maxima is harvested fromthe most external portion of a giant solar evapo-rator of spiral shape (hence the name caracol,meaning snail in Spanish) with a diameter of 3km and a surface area of 900 ha (40) (Fig. 5). Thebiomass is recovered by filtration and, afterhomogenization and pasteurization, spray dried.Daily production of the plant has been reportedto approach 2 tons (dry weight), with a yield of28 tons of protein/ha per year (140). Althoughthe long-term goal is that of a protein source forhuman consumption, so far it appears that S.maxima biomass is commercialized mostly as afeed for animals (including some fancy uses such

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568 CIFERRI

FIG. 5. Plant for the production of S. maxima onLake Texcoco. S. maxima is harvested from theexternal portion of the 900-ha solar evaporator (cara-col) built on Lake Texcoco, Mexico. (Photo courtesyof Sosa Texcoco S.A.)

as to enhance the color of certain Japaneseornamental fish) or as a health food. No datahave been published on the other microorga-nisms present in the waters of Lake Texcoco,how the culture is accomplished, or which nutri-ent is added to enhance growth. It seems likelythat a source of nitrogen (probably nitrate) andphosphorus might be added to achieve rapidgrowth unless these nutrients are supplied byseepage of effluents into the lake. The latterpossibility would explain the high bacterialcounts, including the presence of fecal strepto-cocci, reported for samples of S. maxima fromLake Texcoco (76) and would justify the pas-teurization step involved in the production of S.maxima biomass.

Extensive investigations have been conductedin Israel on the possibility of large-scale cultiva-tion of S. platensis, utilizing brackish waterunsuitable for human consumption or agricultur-al use. In the Negev desert, the cultures aregrown in shallow (0.2-m), black plastic-linedchannels 50 to 300 m long. The channels runback and forth to make, eventually, ponds of 5to 10 ha. To ensure mixing of the culture, thechannels are dug with a constant slope thatprovides a flow of ca. 30 ml/min. At the end ofthe channel maze, the culture is pumped back tothe highest (starting) point (130). Turbulence hasbeen found to be essential to increase growthrate, especially at high population density, sinceit increases the proportion of cells receivinglight. At the high cell concentrations necessaryfor industrial production, if the culture is notagitated only the upper 3 cm of the pond,containing about 20% of the cell population,receives light. In addition to its effects on photo-synthetic efficiency, the occurrence of a "light/dark" cycle seems to be beneficial for growth.Thus, at any cell density agitation increases

output rate, and at optimal ratios of turbulence/population density it may double production(132). Temperature may severely limit growth inwinter months: in open ponds in the Negevdesert, practically no S. platensis was producedfrom December to February (131). The arrest ofgrowth during the winter season was found to bedue to insufficient temperature during the day(average temperature of 18°C compared to val-ues close to 40°C in summer) rather than to thecold temperature of the nights (5°C and, attimes, even 0WC). If the day temperature wasartificially raised from 18 to 25°C, the growthrate was similar to that obtained during thesummer months even when the night tempera-ture approached 0°C. On the other hand, raisingthe night temperature to ca. 10°C did not en-hance growth if the day temperature was notincreased. During the summer months, the limit-ing factor was found to be solar irradiance, andpeak productivity was reached when light levelwas highest. In spring and fall both light andtemperature appeared to limit growth. Examina-tion of productivity in open ponds over a 2-yearperiod demonstrated that growth increased pro-portionally with an increase in temperature andsolar irradiance. Indeed, at any given light inten-sity, growth increased on increasing the tem-perature and similarly, for any temperaturerange, an increase in light resulted in growthincrease (172). Low temperatures, such as thosecommon in winter, play a role also in the mainte-nance of culture purity in open ponds. In wintermonths, when growth of S. platensis was se-verely reduced, a population of Chlorella vul-garis appeared in the ponds reaching up to 50%of the biomass. The increase of the C. vulgarispopulation, as well as of its grazers and theirpredators, could be controlled by raising thepond temperature. Simply covering the pondswith polyethylene sheets raised the temperature5 to 7°C, and this led to a threefold increase inthe S. platensis population with a concomitantdecrease in that of C. vulgaris (from 35 to 40% ofthe total cell volume to <5% [173]). Laboratoryexperiments revealed that another factor, name-ly, the concentration of bicarbonate and gaseousC02, may play a major role in the relativeproportion of C. vulgaris and S. platensis in amixed culture. High concentrations of bicarbon-ate (0.1 M upward) favored growth of S. platen-sis, whereas at low bicarbonate concentrationsand in the presence of CO2 C. vulgaris wasfavored. The effects were specific and not due todifferences in the osmotic pressure or the pH ofthe medium resulting from the removal of bicar-bonate or C02, respectively. Thus, it appearedpossible to maintain a culture of S. platensiswithout significant contamination by the faster-growing C. vulgaris simply by maintaining in the

MICROBIOL. REV.


medium a high concentration of carbonates anda low concentration of CO2 (129). Alternatively,it was possible, at least in a chemostat, tocontrol the contamination of an S. maxima cul-ture by an unidentified species of Chlorella byrecycling part of the biomass recovered afterfiltration through a nylon screen that retainsonly the cyanobacterial filaments (175a).For the Negev area, daily productions of 40

and 10 g (dry weight)/m2 for the summer andwinter months, respectively, have been obtainedto give a yearly production exceeding 62 tons(dry weight)/ha. In warmer climates, such as atArava in the Rift Valley of southern Israel,where year-round operation of the plants seemsfeasible, yearly production may reach 74 tons(dry weight)/ha (11, 130). In Florence, in thecentral part of Italy, experiments performed insmall (up to 100 m2), open ponds have givendaily yields, in the peak summer months, of 14 g(dry weight)/m2. However, due to the fairlycontinental weather, production lasts only 5 to 6months, giving a yearly yield of S. platensis orS. maxima not exceeding 18 to 22 tons (dryweight)/ha (100). The authors have calculatedthat in the southern part of the country, up to300 days of production per year could be ob-tained, increasing productivity to ca. 30 tons(dry weight)/ha. An interesting alternative is thatof growing S. platensis and S. maxima in poly-ethylene tubes 0.3 cm thick and with a diameterof 14 cm (37). The tubes are arranged in a"raceway" fashion and the culture is pumpedthrough the tubes. The advantage of the systemis that the tubes function as solar collectors, thusincreasing the culture temperature in the sea-sons when the atmospheric temperature is toolow to allow growth of Spirulina in open ponds.Daily productivity in tubes has reached 15 g (dryweight)/m2 in summer and 10 g (dry weight)/m2in winter, to give a yearly production estimated,perhaps a bit optimistically, to be ca. 40 to 50tons/ha. Other advantages of the tubular systemare the considerable reduction of water loss byevaporation, the possibility of utilizing sloping(up to 10%) terrains, and the screening fromexternal contaminants (biological or otherwise).In the peak summer season, however, tempera-tures may exceed the limits (40 to 45°C) tolerat-ed by Spirulina and the tubes must either beshaded or cooled with water. In addition, asalready mentioned above, the quality of thebiomass produced in tubes, in terms of proteincontent, seems to be lower than that of thebiomass produced in open ponds.A species of Spirulina, probably S. platensis,

is being considered for mass culture in Taiwan,possibly as a substitute for the too expensivecultures of Chlorella (147). Good results havebeen reported in preliminary trials on the utiliza-

tion of the biomass as food for fish and shrimp.However, the reported economic difficulties en-countered in the production of Chlorella, whichwas mostly sold as a health food in Japan, raiseserious doubts about the financial viability of theproduction of Spirulina as a feed.

Small trials for the production of Spirulina tobe used as a feed or food have been performedor are under way in different parts of the world(10, 40, 168).An attractive possibility is the growth of Spir-

ulina on wastewaters to couple protein produc-tion to recycling of nutrients, removal of organicand inorganic pollutants, and disposal of wastes.Laboratory or small-scale experiments havebeen performed on the growth of S. maxima andS. platensis on city wastewaters (67, 142), cowmanure (108, 145), or swine wastes (26). Inaddition, to reduce the costs of the nutrients tobe added to the medium, attempts are under wayto grow S. platensis by utilizing a variety ofcheap and easily available (especially at thevillage level) substrates such as manure, bone-meal, animal blood, and other wastes (10, 121,146, 149). An interesting possibility is the pro-duction, in rural areas, of carbon dioxide-en-riched air for growth of Spirulina by dung com-posting ("aerobic biogas") (169). All authorsreport encouraging results, but so far no infor-mation about large plants or practical applica-tions have been reported. It is quite likely that inmedia containing organic sources the yields maygreatly surpass those obtained in simple, inor-ganic media (111) but the economics of theprocess, requiring sterilization of the biomassbefore its use even for animal consumption, maystill be unfavorable. Nevertheless, it is fairlyobvious that, in the long run, accomplishment oftwo goals, recycling of industrial or urban waste-waters plus production of protein biomass,makes such processes very attractive. One canenvisage cultures of Spirulina in "clean" waterswith chemically defined media to produce bio-masses for alimentary consumption and culturesin "dirty" waters to give biomasses to be usedas a feed or as a starting material for the extrac-tion of chemicals.

NUTRITION AND TOXICOLOGYSeveral investigations have been performed

on the possible utilization of S. platensis or S.maxima as a food source for human or animalconsumption. Such investigations were stimulat-ed by the discovery of the high protein contentof Spirlulina biomasses and by reports indicatingits use as an aliment by some populations of theChad area and, possibly, by the inhabitants ofMexico before the Spanish conquest. Further,the blooms of Spiriilina have always represented

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FIG. 6. Lesser flamingos feeding on S. platensis.Aerial photo of a flock of lesser flamingos (P. minor)feeding on S. platensis along the north shore of LakeNakuru (Kenya) in 1973. Over 90% of the birds in theflock congregate close to shoreline where wind in-duces the formation of high-density mats of S. platen-sis. (Photo by E. Vareschi; reprinted from reference166 with permission of the publisher.)

a major, if not the only, source of food for birdsin the areas where Spirulina is the predominantcomponent of the phytoplankton. Indeed, allobservers, from the Spanish chroniclers of themiddle ages (34) to today's ecologists (79, 156,166), have recorded the abundance of avifaunain lakes containing Spirulina, especially in theperiods in which the cyanobacterium is moreabundant (Fig. 6). Analysis of the stomach con-tent of the lesser flamingos (Phoeniconaias mi-nor), the most numerous birds of the Rift Valleylakes, showed that, at least during the periods inwhich the collections were made, the birds were

feeding entirely on Spirulina (128, 133). Forsome lakes of the same area, Tuite correlatedthe presence of lesser flamingos with that ofhigh-density blooms of the cyanobacterium;when the Spirulina blooms were abundant, thebirds fed on the lakes and stayed in the area tobreed (156). If, however, the population of Spir-ulina decreased, as has happened in some of theRift Valley lakes after a change in the concentra-tion of chemicals in the waters, then the birdswere compelled to rely on other cyanobacteriaor on benthic diatoms. Since the lesser flamin-

gos have apparently evolved a filter-feeding ap-paratus (80) that is very efficient for Spirulinaand much less so for the unicellular forms (Fig.7), the disappearance or the reduction of theSpirulina population causes dispersing of thebird flocks to other bodies of water in easternand southern Africa. Thus, when, in 1973 to1974, a dramatic decrease in the Spirulina popu-lation occurred in Lakes Nakuru and Bogoria,the flamingo population almost disappearedfrom these lakes. When, 5 years later, due toheavy rainfall, the concentration of chemicalschanged again in these two lakes, the populationof Spirulina rose rapidly at least in Lake Bo-goria, resulting in an almost immediate return ofa dense and stable population of flamingos. ForLake Nakuru, Vareschi has recorded the quanti-tative variations of the cyanobacterial and algalpopulations for almost 10 years (1972 to 1980)and the associated variations in the flamingoflocks (166, 167). S. platensis, which accountedfor almost 100% of the biomass at the beginningof 1973, began to decline so that, at the end ofthat year, it represented <1% of the phytoplank-ton. Up to 1980 this cyanobacterium was presentbut did not constitute a significant proportion ofthe phytoplankton. Concomitant with the de-cline in S. platensis population, another unclas-sified species of Spirulina became the mostabundant species in the years 1974 and 1975. Inturn, this species too declined in abundance, andby 1977 the lake presented a mixed population ofAnabaenopsis, single-cell cyanobacteria, greenalgae, and diatoms. These changes were proba-bly brought about by an increase in the watersalinity which favored growth of other organ-isms while reducing that of Spirulina and that ofthe zooplankton population responsible for mostof the nutrient recycling. These factors, possiblyassociated with others such as a postulatedappearance of cyanophages specific for Spiru-lina or its selective photooxidation, were re-sponsible for the practical disappearance ofSpirulina and the associated population of lesserflamingos that decreased from ca. 106 in 1973 tojust several thousand.

Evaluation of the nutritional characteristics invitro confirmed that drum-dried S. platensis orS. maxima represents a valuable source of ali-mentary protein (93). Although, as already men-tioned, the content of methionine+cysteine andlysine of Spirulina protein is somewhat lowerthan that of reference protein sources such aslactalbumin or the Food and Agricultural Orga-nization protein pattern, in vitro studies indicat-ed that the cyanobacterial protein is nutritionallysuperior to legume protein, although inferior tomeat protein. A number of nutritional studieshave been performed on different animals (mice,rats, pigs, chicken, calves) fed diets in which

MICROBIOL. REV.


B

FIG. 7. Filter-feeding apparatus of lesser flamin-gos. The platelets of the filter-feeding device in the billof lesser flamingos (P. minor) appear to be welladapted to trap filaments of S. platensis (A) ratherthan unicellular cyanobacteria (B). (Photo by E.Vareschi; reprinted from reference 166 with permis-sion of the publisher.)

Spirulina was substituted totally or in part forthe protein requirement. By and large, S. maxi-ma or S. platensis, either drum or spray dried,was well accepted by animals, giving, in gener-al, weight increases and nitrogen deposition inthe body comparable to, if not better than, thoseobtained with most other plant protein sources(10, 12-14, 26, 45, 170). No toxic effect orabnormality on postmortem observation wasreported in these experiments or in long-term(18-month) feeding trials (15) or short-term mas-sive feeding trials in which up to 800 mg/kg ofbody weight was administered orally for 12 days(88). In addition, negative results were reportedin a multigeneration study in mice (121) and inmutagenicity tests with Salmonella typhimuriumand Schizosaccharomyces pombe performed onurines of animals fed Spirulina for 4 months (12).Careful evaluation in rats of several parameterssuch as increase in body weight, total bodynitrogen, and levels of serum total protein andalbumin led to the conclusion that S. platensisand S. maxima represent protein sources asgood as legumes, including soybeans, but inferi-or to the best protein source, lactalbumin (78). Asimilar conclusion was reached by assaying pro-tein synthesis in vitro by ribosomes isolatedfrom skeletal muscles of rats fed with differentprotein sources (107). The assay, which is posi-tively correlated with the quality of alimentaryprotein and hence is a measure of the nutritionalvalue of a protein source, revealed that thenutritional quality of S. platensis was acceptablealthough lower than that of casein plus methio-nine. If methionine was added to the Spirulinadiet, its nutritional quality improved, although itnever reached that of casein and methionine.Similar conclusions were reached for differentpreparations of Saccharomyces cerevisiae, themost typical source of SCP. In hens, administra-tion of Spirulina slightly stimulated egg produc-tion but not the eggs' size, whereas it greatlyincreased yolk color (10, 13). In young shrimps,prawns, and fries of different fish of commercialimportance, Spirulina sustained increases inbody weight and length as well, if not better,than standard diets. In addition, for a number ofspecies, sexual maturity was reached earlier,thus allowing shorter breeding cycles. Finally,Spirulina-fed fish acquired a pink-yellow pig-mentation of the meat which appears to be ofcertain importance from a commercial point ofview.Becker (9) has summarized the standard pa-

rameters used to evaluate the nutritional qualityof a protein source. The values reported for S.platensis are lower than those of a standardprotein, casein, but similar to those found forScenedesmus acutus and superior to those re-ported for two other algae under test (Table 4).

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TABLE 4. Nutritive value of S. platensis and various algae"

Algae Protein efficiency Net protein Biological Digestibilityratio' utilization' value" coefficient'

Spirulina platensis 1.80 62.0 75.0 83.0Scenedesmius acutus 1.93 65.8 80.8 81.4Uronema sp. 1.43 46.0 55.0 82.0Coelastrum sp. 1.68 57.1 76.0 75.1Casein (standard) 2.50 83.4 87.8 95.1

" Data from reference 9.b Calculated from the gain in body weight and protein consumption.' Percentage of N consumed which is retained in the body.d Ratio of N adsorbed to total N intake.e Proportion of food N absorbed.

There are few and incomplete reports of ex-periments on humans. Pirie (119) quotes experi-ments performed in Mexico in which 20 to 40 gof dried S. maxima was given daily to athletesfor periods ranging from 30 to 45 days "withgood results." Similarly, reports of favorableresults obtained in the case of Mexican childrenor infants suffering from severe malnutritionhave been cited (27, 40). In a more detailedstudy, diets in which up to 50% of the proteincame from S. maxima were fed through a plastictube to five undernourished adults for periodsvarying from 4 to 5 days (141). A significantweight gain and a positive nitrogen balance wereobserved and no side effects were reported. Ofthe other biological parameters evaluated, only amodest increase in the serum, but not in urinary,uric acid was noticed.A careful evaluation of the consumption of S.

platensis in the Chad area was reported in 1976(33). It appears that the area in which Dihe iseaten regularly is restricted to a fairly limitedregion, east and northeast of Lake Chad, with atotal population of ca. 300,000. Of this popula-tion only the quantitatively most important eth-nic group, the Kanenbou, consumes Dihe regu-larly, whereas its consumption is nil among thefishermen living around the lake and the nomadsnorth of it. Among the Kanenbou, Dihe is eatenfrequently; depending on the season, Dihe ispresent in 7 of 10 meals. Direct consumption ofthe Dihe biscuits takes place only for supersti-tious reasons among pregnant women becauseof the belief that its dark color will screen theunborn baby from the eyes of sorcerers. Ingeneral, Dihe is eaten as a constituent of anumber of sauces that always accompany thestandard millet meal. The dry Dihe is pounded ina mortar and the powder is suspended in water.Salt, pimento, tomatoes, and, if available,beans, meat, or fish are added to complete thesauce. In a meal, a person eats approximately 10to 12 g of Dihe which satisfies at most 8% of thecaloric need and little more than 10% of the

protein requirement. That Dihe may representan "emergency" sort of food may be inferredfrom the finding that its consumption decreaseswhen the economic conditions, or the localavailability, allow consumption of meat or fish.However, during periods of severe famine, Diheis still consumed extensively although one ex-pects that these periods may often be the resultof severe droughts that may cause drying ofmany temporary lakes, thus reducing the supplyof Spirulina. No information is available toindicate if, in the past, Dihe represented a quan-titatively more important source of food or if itsconsumption was geographically more wide-spread than today. It was pointed out that al-though certain lakes, such as that of the oasis ofOuniangakebir, 1,000 km northeast of LakeChad, contain a population of S. platensis asabundant as that of Lakes Rombou and Bodou,no record was found for a local consumption ofDihe (91). Yet, it seems rather unlikely that,during periods of famine, the information aboutthe presence in the lakes of an easily availablefood source should not have arrived from theneighboring populations.

CONCLUSIONS AND PROSPECTS OF THEUTILIZATION OF SPIRULINA AS A FOOD

SOURCEFirst, one may ask whether there is any sense

today in looking for nonconventional sources ofprotein such as SCP for animal and, possibly,human nutrition after the failure in the last 2decades in developing its production, at least inwestern countries. Second, but perhaps evenpreliminary, is the question: does the so-calledprotein gap in human nutrition that polarized theattention of nutritionists, food scientists, andagronomists on the production of protein-richfoodstuff still exist? If the answer to these twoquestions is affirmative, one may then askwhether Spirulina has characteristics both in-trinsic (e.g., chemical composition, toxicity) and

572 CIFERRI MICROBIOL. REV.


extrinsic (e.g., production technology, yield)which justify singling out this organism amongall those recognized as possible sources of foodor feed.

Concerning the first question, it must be con-sidered that even today starvation and malnutri-tion are still widespread in vast portions of theworld and that, notwithstanding the success ofthe various "green revolutions," present-dayagriculture seems incapable of satisfying themost basic human need, adequate nutrition. Theproduction of microorganisms to be used as foodor feed has evident advantages. It does notcompete with conventional agriculture for land,it is less dependent on favorable weather condi-tions, and its yields, in terms of surface or time,are much higher than those of agriculture. Thenegative outcome of the attempts at SCP pro-duction (mostly yeasts on hydrocarbons) in the1960s and 1970s in western countries was theresult of the increase in petroleum costs and ofthe negative reaction of the potential consumersafraid, rightly or wrongly, of the possible detri-mental effects on health associated with thedirect consumption of SCP or even of meatsfrom animals fed these biomasses. Yet produc-tion and use of SCP in the form of yeasts grownon petroleum derivatives are increasing in othercountries. Indeed, for instance, in the U.S.S.R.its annual production was approaching 5 x 105tons already in 1977 (90a). (Ironically, it ispossible that some of the countries that havebanned the use as a feed of petroleum-grownSCP regularly import meat from animals fed thistype of SCP.) Thus, production of microbialbiomasses other than those grown on petroleumderivatives is still conceivable, as demonstrated,for instance, by the British development in the1980s of the mass production on methanol of thebacterium Methylophillus methylotrophlus.Even if the protein gap is now a bit passe (117,

179), there is no doubt that one of the deleteri-ous effects of undernutrition is due to insuffi-cient protein ingestion. It appears that proteindeficiency, especially in very early life includingthat before birth, results in serious and irrevers-ible damages to body development and mentalhealth. Yet, the typical crops of the world areaswhere starvation or malnutrition occur regularlyare energy rich but protein insufficient (rice,cassava, wheat, etc.) (77). Thus, there appear tobe regions of the world in which calorie intakemay be sufficient but physical and mental devel-opment, especially of children, is impaired bylack of an adequate protein supply. In manyareas of Asia, for instance, over the last 2decades rice and wheat production have in-creased significantly but production of legumes,a source of protein complementary to that ofcereals, has fallen drastically (67). Therefore, it

still appears desirable to produce protein to beused, directly or indirectly, for human consump-tion in a way that complements, rather thancompetes with, traditional agriculture.

In this context, therefore, the possible exploi-tation of Spiriulina as a source of protein must belooked at in the light of the following consider-ations.

(i) Natural alkaline lakes, in which S. platensisand S. maxima grow abundantly if not exclu-sively, are usually found in arid areas of thetropics and subtropics where malnutrition isoften endemic.

(ii) The requirements of a very alkaline pH ofthe growth medium ensures that carbon dioxideis retained in the waters in contrast to the rapidloss observed at the acid pH required by unicel-lular algae such as Chlorella, Scenedesmlus, andEuglena. The alkalinity of the medium drastical-ly reduces growth of the majority of other micro-organisms, including those pathogenic to hu-mans and other animals.

(iii) The spiral shape of the trichome and thepresence of gas vacuoles result in the formationof floating mats that may be easily harvested bygravity filtration, thus reducing considerably theenergy requirements associated with the recov-ery of single-cell microorganisms.

(iv) S. platensis and S. maxima have an ex-tremely high protein content (up to 70% of thedry weight), thus representing one of the richestprotein sources of plant origin. The proteinquality is among the best in the plant world andany amino acid unbalance may be easily correct-ed.

(v) Like all other microbial cells, Spiriulinacontains vitamins and growth factors, but its y-linolenic acid content, a candidate growth factorfor humans, is the highest after milk and the oilof the evening primrose (Oenothera biennis).

(vi) The concentration of nucleic acids isamong the lowest recorded for microbial cetlsconsidered for use as food or feed.

(vii) Spirlulina cells are enclosed by a thintrichome sheath and by a murein-containing cellwall which make Spiruilina protein more easilydigested by animals than that in yeasts andunicellular algae (7).

(viii) Yields per unit area are, at least in thelaboratory and small pilot plants, spectacular,and these figures become even more impressiveif expressed in terms of protein yield (Table 5).Thus, it has been calculated that the amount ofland necessary to satisfy the yearly proteinrequirement of a human is ca. 5 ha for meat fromcattle on grassland, slightly less than 1 ha in thecase of wheat, and approximately 10 m2 in thecase of S. platensis (171). Although the produc-tion figures have not been tested in large-scaleindustrial plants, it must be remembered that

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TABLE 5. Yields of traditional crops' and ofcultures of Spirulina

Crop Total yield Protein Protein yieldCrop (tons/ha per yr) content (%) (tons/ha per yr)

Wheat 6.7 9.5 0.64Maize 14 7.4 1.04Rice (hulled) 8 7.1 0.57Soybeans 4 35 1.4S. platensisb 60-70 65 39-45S. maxima" 40 70 28

a Data from reference 11.b Estimated for production plants in Israel (11, 130).' Calculated for Lake Texcoco (40).

nothing so far has been done to improve thestrains or the culture conditions in terms ofmedium, plant design, or operation. In addition,cultures may be performed on marginal landunsuitable for conventional agriculture and uti-lizing industrial surpluses such as CO2 from fuelcombustion, warm waters from cooling plants,etc.

(ix) As for all photosynthetic organisms, thegrowth requirements of Spirulina are minimal.However, compared with traditional crops, cul-tures in open systems of Spirulina and of othermicroorganisms as well have a very high waterrequirement. Especially in tropical or subtropi-cal climates, evaporation causes a significantloss of water that may be only in part compen-sated by rainfall. Thus, it has been calculatedthat microalgae, and certainly also Spirulina,grown in open ponds consume more water perunit area (25,000 m3 of water per ha) than evenrice (17,000 m3 of water per ha) (116). Yet, ifwater consumption is calculated in terms of theprotein yield, microalgae have water require-ments (1,000 m3 of water per ton of protein) thatare lower than those of traditional crops, includ-ing soybeans (7,000 m3 of water per ton ofprotein).

(x) Extensive nutritional and toxicologicaltests in a variety of animals indicate that Spiru-lina is a valuable and safe source of protein.From a nutritional point of view, although inferi-or to the best protein of animal origin, Spirulinaprotein ranks as one of the best protein sourcesof plant origin.

(xi) Records exist that, since time immemori-al, Spirulina has been a component of the every-day diet of certain human populations and that itis still the main source offood of some species ofbirds.Development of mass production of Spirulina

depends on the availability of reliable data onthe economics of the process, at least at the pilotplant scale, possibly on year-round operation. Itwould be equally important to evaluate the per-

formance in terms of growth velocity, yield,chemical composition, and susceptibility to con-tamination by other microorganisms of thestrains so far available when utilized on continu-ous or semicontinuous processes. On a labora-tory scale, it may be especially fruitful to searchfor other strains or for the isolation of mutantsendowed with more favorable characteristics(faster growth, better yields in terms of totalbiomass, or of specific cell constituents, capaci-ty to grow at temperatures above or below theoptimal ones for the existing isolates, etc.).Finally, any extensive genetic alteration of thepresently available strains depends on the un-raveling of the mechanism for genetic recombi-nation, if it exists, or the development of othermeans to manipulate Spirulina genetically.

ACKNOWLEDGMENTSThe work performed in my laboratory was supported by

grants from Consiglio Nazionale delle Ricerche and Ministerodella Pubblica Istruzione.

I gratefully acknowledge the many colleagues that havegenerously supplied preprints, photographs, and other materi-al, especially A. Iltis, E. Vareschi, A. Richmond, L. V.Venkataraman, S. Litvak, S. Golubic, R. Locci, E. W.Becker, and R. Materassi. Finally, I am greatly indebted toMaria Gravagna for continuous help in the preparation of themanuscript.

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11. Berend, J., E. Simovitch, and A. Ollian. 1980. Economicaspects of algal animal food production, p. 799-818. InG. Shelef and C. J. Soeder (ed.), Algae biomass. Else-vier/North-Holland Biomedical Press, Amsterdam.

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