Spirulina platensis
MORPHOLOGY & ULTRASTRUCTURE
C VAN EYKELENBURG
Te
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SPIRULINA PLATENSIS
MORPHOLOGY 8 ULTRASTRUCTURE
TR dsss
1252
SPIRUL1NA PLATENSIS
MORPHOLOGY
& ULTRASTRUCTURE
PROEFSCHRIFT
TER VERKRIJGING VAN DE GRAAD VAN DOCTOR
IN DE TECHNISCHE WETENSCHAPPEN AAN DE
TECHNISCHE HOGESCHOOL DELFT, OP GEZAG
VAN DE RECTOR MAGNIFICUS PROF. IR.
B.P.TH. VELTMAN, VOOR EEN COMMISSIE
AANGEWEZEN DOOR HET COLLEGE VAN DEKANEN
TE VERDEDIGEN OP DONDERDAG 20 NOVEMBER
1980 TE 16.00 UUR
DOOR
CAROLUS VAN EYKELENBURG
SCHEIKUNDIG INGENIEUR
GEBOREN TE ROTTERDAM
1980
IS GOEDGEKEURD DOOR DE PROMOTOR
DIT PROEFSCHRIFT VJERD BEWERKT OP HET LABORATORIUM VOOR. ALGEMENE
EN TOEGEPASTE MICROBIOLOGIE EN HET LABORATORIUM VOOR ALGEMENE
EN TECHNISCHE BIOLOGIE VAN DE TECHNISCHE HOGESCHOOL DELFT
VOORWOORD
Mijn dank gaat uit naar een ieder die op enigerlei wijze heeft bijgedragen
aan de totstandkoming van dit proefschrift of een onderdeel daarvan.
(Urn zu erkennen, ob das Bild wahr ader falsch ist,
mussen wir es mit der Wirklichkeit vergleichen.
Nur SO könnten wir a priori wissen, dass ein
Gedanke wahr ist, wenn aus dem Geênnken selbst
(ohne Vergleichsobdekt) seine Wahrheit zu
erkennen ware.
Satze 2.223 und 3.05 Tractatus logico-philosophieus.
Wittgenstein, L, J. J. 1921. Logisch Philosophische
Abhandtung. Annalen der Naturphilosophie 14: 184-262.)
C O N T E N T S
A P P E N D I X TO III
INTRODUCTION TO IV
IV RAPID R E V E R S I B L E M A C R O M O R P H O L O G I C A L CHANGES IN
SPIRULIHA PLATEI1SIS
(published in Naturwissenschaften 67 (1980) 200-201)
I G E N E R A L INTRODUCTION 7
HISTORY
REMARKS ON TAXONOMY
SPIRULIHA AS A SOURCE OF NUTRITIONAL PROTEIN
A I M OF THIS INVESTIGATION
REFERENCES
INTRODUCTION TO II AND III
I I ON THE MORPHOLOGY AND ULTRASTRUCTURE OF THE CELL
WALL OF SPIEVLINA PLATEHSIS
( p u b l i s h e d i n A n t o n i e van Leeuwenhoek 43 (1977) 89-99)
I I I SOME THEORETICAL CONSIDERATIONS ON THE IS VITRO
SHAPE OF THE CROSS-WALLS IN SPIRVLIHA s p p .
( p u b l i s h e d i n J . t h e o r . B i o l . 82 (1980) 271-282)
45
51
V A GLUCAN FROM THE CELL WALL OF THE CYANOBACTERIUM
SPIRULIHA FLA TEH SIR
(published in Antonie van Leeuwenhoek 44 (1978) 321-327) 55
INTRODUCTION TO VI 6 3
VI AN 'ECOBOX' WITH A DISCONTINUOUS TEMPERATURE GRA
DIENT AND A CONTINUOUS LIGHT INTENSITY GRADIENT
(published in Experientia 35 (1979) 1127-1123) 65
INTRODUCTION TO VII 69
VII THE U L T R A S T R U C T U R E OF
SPIRULIHA PLATENSIS IN RELA
TION TO T E M P E R A T U R E AND LIGHT INTENSITY
(published in Antonie van Leeuwenhoek 45 (1979) 369-390) 71
INTRODUCTION TO VIII 93
VIII ECOPHYSIOLOGICAL STUDIES ON
SPIRULIHA PLATEHSIS.
EFFECT OF T E M P E R A T U R E , LIGHT INTENSITY AND N I T R A T E
CONCENTRATION ON GROWTH A N D U L T R A S T R U C T U R E
(published in Antonie van Leeuwenhoek 46 (1980) 113-127) 97
SUMMARY AND DISCUSSION 113
I . G E N E R A L I N T R O D U C T I O N
7
H I S T O R Y £
C y a n o b a c t e r i a have been p r e s e n t on the surface of the Earth since the early 9
Precambrian about 3.6 10 years ago (Stewart, 1977; Ford, 1980) . F i l a m e n t o u s , c y l i n d r i c a l , u n b r a n c h e d , smooth and u n l a m e H a t e d forms with a diameter up to 10 urn a.'"! at least 25 times longer than wide are found in fossils from late P r e
-9
Cambrian (1.1 10 y e a r s a g o ) , h y p e r s a l i n e lagoons (Oehler, Oehler and S t e w a r t , 1 9 7 9 ) .
In his second report to Spain (1520) Hernan Cortes (14851547) mentions t e -c u i t l a t l , a p r o d u -c t whi-ch was eaten in -considerable amounts by the pre--conquest inhabitants of T e n o c h t i t l a n (residence of t h e high p r i e s t Tenoch) (the p r e s e n t Mexico C i t y ) , and w h i c h the Spaniards also found p a l a t a b l e . At the time of the conquest the local p o p u l a t i o n in this area has been estimated to number 2 50,000, the feeding o f w h i c h w o u l d exceed the capacity of the low levels of cattle breeding and a g r i c u l t u r e p r a c t i s e d . It is considered likely that tecuitlatl
(Deevey, 19 57) made from a c y a n o b a c t e r i u m (Ancona, 1933) found in Lake Texcoco made u p the s t a p l e p a r t of t h e n a t i v e s d i e t . In the sixteenth century this lake w a s twenty t i m e s larger than it is today (Ortega, 1 9 7 2 ) , and the cyanobacterium growing in it could w e l l h a v e provided the major source of p r o t e i n .
T h e n a t u r a l i s t F r a n c i s c o H e r n a n d e z ( 1 5 1 3 - 1 5 8 7 ) , sent by the Council of the Indies to report on the f l o r a , fauna and minerals of New S p a i n , was probably the first to give scientific information on the cyanobacteria w h i c h h e , erroneously, thought to b e a m i n e r a l . His report w a s compiled around 1550-1560, but the m a nuscripts w e r e scattered and lost. In 1790, they w e r e published incomplete
Since 1971, the name blue-green alga is gradually b e i n g replaced in the l i t e rature by c y a n o b a c t e r i u m (Stanier et a l . , 1971, see also C o h n , 1 8 5 3 ) ; in 1978 a proposal w a s p u t forward (Gibbons and Murray, 1978) to validate the Cyanobacte-vidtes as a new order of the kingdom of the Pvocar-yotae, while Stanier et a l .
(1978) p r o p o s e d the p l a c i n g of the nomenclature of the cyanobacteria under the rules of the International Code of Nomenclature of b a c t e r i a . These p r o p o s a l s have been seriously questioned by Bourrelly ( 1 9 7 9 ) , Geitler (1979) and Golubic
8
(F. Hernandi, Opera, cum edita, tum inedita, Madrid, 1790). In his 'Essai
Poli-tique sur Ie Royaume de la Nouvelle Espagne' (1811) , Von Humboldt refers to
Bernardino de Sahagun who, about 1550, described the clear blue colour of
tecuit-latl In his Universal History (Parrar, 1966, Ortega, 1972). We now know from the
tecuitlatl still sold in the area that the cyanobacterium concerned is
Spivulina
platensis. It is an obligate photo-autotrophic organism, having an absolute re
quirement for light and there is no stimulation of growth or respiratory acti
vity by reduced carbon compounds (Carr, 1979).
Reports on the presence of
Spivulina in Africa date back to 1896 when west
and West recorded the cyanobacterium in a collection taken from Lake Losuguta
in Kenya. Jenkin (1929) , who participated in the Percy Sladen Expedition to East
Africa, collected and described the cyanobacterium from lakes Baringo, Naivasha,
Nakuru and Elmenteita. Rich (1931, 1933) published an account on the
phytoplank-ton collected from the lakes of Kenya and Uganda and confirmed the findings of
Jenkin (1929). Ross (1953) described the water in the Ferguson Gulf of Lake Ru
dolph as having the appearance of green soup, due to a very thick population of
Spivulina and Anabaenopsis.
Dangeard (1940) was the first to describe an edible cyanobacterium, collected
and eaten by man in Central Africa. It proved to be a mass of helical filaments
of a cyanobacterium now known to be
Spivulina plateneis.
During the winter of 1964-1965, members of the Belgian Sahara Expedition pur
chased , in the market of Fort Lamy near Lake Chad, flat cakes of a greenish edi
ble substance called dihé. These appeared to consist solely of a cyanobacterium
collected from the bottoms of seasonally dried up ponds in the North of Lake
Chad and consumed by the local population. In the region of Ounianga Kébir,
about 750 miles northeast of Fort Lamy, the members of the expedition were
struck by the abundance of a microscopic alga in some lakes. Compere of the
State Botanical Garden in Brussels examined the product and found it to consist
solely of a cyanobacterium:
Spivulina platensis (Leonard, 1966, Leonard and
Compere, 1967). From chemical analysis carried out at the Laboratoire
Intercom-munal de Chimie et de Bacteriologie in Brussels it appeared to be very rich in
protein and therefore very useful for consumption by the protein-deficient
desert nomads. These findings drew considerable attention and led to investiga
tions on the organism concerned.
Outside Africa and Mexico,
Spivulina platensis appears to be less common. In
Asia it has been harvested at Lahore (Pakistan) (Ghose, 1923; Rhandawa, 1936),
Calcutta (India) (Biswas, 1927) and in Lake Beira (Sri-Lanka) (Holsinger, 1955)
but never in the abundance seen in the African lakes. None of the authors who
studied the organism in Asia mentioned human consumption.
9
The
Spivulina platensis strain used in the present study originates from
Lake Nakuru, a shallow soda lake in the Kenyan part of the African Rift Valley.
The lake has no surface outlet and the level fluctuates in response to rainfall
2
and evaporation. The surface area is about 35 km when the lake is full. The
electrical conductivity of the lake water is high (15-30 mS/cm at 20 C) and the
pH varies from 9 to 11. There is a very high photosynthetic activity with a net
primary production of 900-1800 tons fresh weight per day.
Spivulina platensis is
the dominating species and serves as the main source of food for the huge flocks
of lesser flamingos
(Phoeniconaias minor) for which the lake is famous
(Kallq-vist and Meadows, 1978; Vareschi, 1978).
REMARKS ON TAXONOMY
The taxonomy of the cyanobacteria, or blue-green algae, has always caused frus
tration. Wallroth (1833) called the group
Myxophykae but Stitzenberger (1860)
changed this to
hfyxophyceae,- Rabenhorst (1863) proposed
'Phycochvomophyo.es
,
while Sachs (1874) called them
Cyanophyceae, a name which Kirchner (1878) with
out success, tried to change to
Schizophyeeae.
The distinction within the cyanobacteria, between the genera
Spivulina
(Tur-pin, 1827) and
Avthvospiva (Stitzenberger, 1852) has long been subject of con
troversy . Traditionally, members of the
Oscillatoviaceae lacking a sheath but
with regularly helical (spiralized) septate trichomes have been placed in the
genus
Avthvospiva, and those with non-septate regularly helical trichomes have
been placed in the genus
Spivulina (Smith, 1950; Prescott, 1951). Generic sepa
ration based on the presence or absence of cross-walls has been questioned by
numerous workers dating back to early in the 20 century. Schmid (1920) and
Figini (1925), working with species then described as
Spivulina, showed that in
many species septa are demonstrated after prolonged staining with neutral red.
As a result, there was a tendency to place species of
Avthvospiva into Spivuli
na, since the name Spivulina has priority over that of Avthvospiva. However,
Crow (1927) maintained that certain species were non-septate and suggested that
Spivulina should be reserved for such species, the septate forms being placed
in the
Avthvospiva group. Such has been the practice of many authors, particu
larly in the USA. Using a special staining technique Gorbunova (1958) was able
to show the presence of septa in
Spivulina majov, Kütz., a species commonly used
to demonstrate the non-septate nature of typical
Spivulina species, and on this
basis again recommended combining
Avthvospiva and Spivulina. Holmgren, Hostetter
and Scholes (1971) proposed an investigation of all species concerned in order
to establish the presence or absence of septa, but this was not followed up.
10
Geitler (1932) did not recognize the generic distinction between
Arthro&pira
and
Spirulina. Iltis (1970) integrated the genus Spirulina (Arthrospira) into
the genus
Oscillatoria without any result.
In a paper on generic assignments, strain histories and properties covering
150 genera and well over 1000 species, Rippka et al. (1979) state that substan
tial differences in DNA base composition suggest a more solid genetic basis for
recognizing two genera in the future. The generic subdivision of the filamen
tous , non-heterocystous cyanobacteria assigned to Section III (filamentous
cya-nobacteria; a trichome which grows by intercalary cell division; reproduction
by random trichome breakage, by formation of hormogonia; a trichome always com
posed only of vegetative cells,- division in only one plane) in the terminology
of Rippka et al. (1979) provides three genera:
Oscillatoria, Pseudoanabaena and
Spiruliiia which can be distinguished on structural grounds. As stated by Herdman
et al. (1979a), a clear-cut generic assignment for many of the strains cannot
yet be made since these strains share, in various combinations, the properties
which have been ascribed to the genera
Lyngbya, Vleotonema and Phormidium. These
have been placed in a provisional category, termed the LPP group (see Rippka
et al., 1979).
Oscillatoria and Pseudoanabaena have relatively narrow and simi
lar base compositional spans of 40 to 50 and 44 to 52 mol % GC, respectively.
The two strains of
Spirulina analysed by Herdman et al. (1979a) had DNA with 44
and 54 mol % GC. These two strains differ greatly in phenotypic respect: strain
PCC (Pasteur Culture Collection) 7345
(Arthrospira platens-is) contains gas va~
cuoles and forms very thick filaments up to 16 um wide, whereas strain PCC 6313
(Kenyon, Rippka and Stanier, 1972) does not form gas vacuoles and has much thin
ner filaments (Herdman et al., 1979a'and Rippka et al., 1979).
According to Herdman et al, (1979b),
Spirulina differs from the majority of
the Section III organisms (Rippka et al., 1979) in the possession of a small
9 9
genome of 2.53 10 dalton, whereas the average size for Section III is 3.79 10
dalton with respect to genome size, while this Section is well separated from
the LPP group.
SPIRULIIIA -AS A SOURCE OF NUTRITIONAL PROTEIN
'Every attempt must be made to inform the scientific community of the increasing
interest, as a potential source of food, in blue-green algae in general and the
genus
Spirulina in particular. Determination of the potential of algal protein
in animal and human nutrition will reguire the examination of many strains,
cultural conditions and processing techniques. In view of the meager knowledge
of fine structure and physiological functions as taxonomical criteria for the
11
blue-green algae, the fundamental knowledge of these organisms must be in
creased'. The statement above is the first paragraph of the 'Conclusions made
at the conference "Preparing nutritional protein from
Spiruli?ia" in Stockholm,
June 13-15, 1968'.
Since 1965, much work has been done on
Spirulina species. Studies have been
published on optimal medium composition (Zarrouk, 1966), on growth (Ogawa and
Terui, 1970; Ogawa, Kozasa and Terui, 1972), on carbon requirement {Ivolgina,
Meshcheryakova and Al'Bitskaya, 1972),
on growth yield in continuous culture
(Aiba and Ogawa, 1977), and on chemical factors influencing growth (Crance,
Forin and Baron, 1977) .
Besides these more technological studies, investigations into the possibili
ties of using
Spirulina for human nutrition have been published. For instance,
Clément, Durand-Chastel and Henny (1969) and Wachowicz and Sagrodzki (1976)
have evaluated the proteins, amino-acid composition and nucleic acid content.
Hedenskog et al. (1969) have investigated methods to increase digestibility.
The variation in lipid composition was studied by Paoletti, Materassi and
Pelo-si (1971) while the mutational effects of ultraviolet rays and antibiotics on
Spirulina platens-is were examined by Pelosi, Pushparaj and Florenzano (1971).
Delpeuch, Joseph and Cavelier (1975) gave detailed information on where and
how dihé is eaten in Chad, they also reported on the nutritional values of
Spi
rulina platensis gathered in different seasons.
The acceptibility of various culinary products based on the alga
Spirulina
was tested by Sautier and Trémolières (1975). They conclude that
Sp-im.ili.na
is
little appreciated in France due to its offensive colour, smell and taste. How
ever, no intestinal problems occurred, and the food did not modify the balances
investigated, although faecal nitrogen increased to 2.08 g as compared with con
trol period values between 1.33 g and 1.51 g when people were provided with 50%
cyanobacterial protein. As the uric acid level in urine did not vary and the
serum values increased only slightly (cf. Feldheim et al., 1973), they conclu
ded that ingestion of
Spivulirux in small doses even over a long period of time
should be tolerable for humans. Boudêne, Collas and Jenkins (1975) could not
find any evident toxicity related to the organism. Very low concentrations
(2 to 3 ppb) of 3,4-benzpyrene in variously prepared dry
Spirulina samples have
been reported by Bories and Tulliez (1975). Jacguet (1975} showed that
Spirulina
preparations are accompanied by other micro-organisms, the species depending on
the method of processing. However, faecal streptococci, Enterobacteriaceae,
yeasts and mould spores are exceptional.
The protein content of dried
Spirulina has been measured in numerous studies
and varies between 60 and 72%. The dried, decolourized product can reach a
pro-12
tein c o n t e n t of 8 4 . 2 % (Baron, 1975) w i t h o u t causing h y g i e n i c or n u t r i t i o n a l in c o n v e n i e n c e . Many s t u d i e s , mainly by R u s s i a n and F r e n c h a u t h o r s , h a v e been p u blished on the n u t r i t i o n a l value of Spirulina for animals (i.e. see Annales dc
la N u t r i t i o n et d e 1'Alimentation volume 2 9 , 1 9 7 5 ) .
It should b e emphasized that for single c e l l p r o t e i n , Spivulina platensis is
most economically grown in areas w i t h h i g h luminous f l u x , a v e r a g e t e m p e r a t u r e s a b o v e 25 C , and in natural w a t e r s such as T e x c o c o Lake near M e x i c o C i t y , A f r i c a n lakes and Indian v i l l a g e p o n d s w h e r e 'low-cost t e c h n o l o g y ' is used for c u l t i v a tion (Seshadri and T h o m a s , 1 9 7 8 , 1 9 7 9 ) .
A I M O F T H I S I N V E S T I G A T I O N
As stated p r e v i o u s l y , Spivulina platensis m i g h t play a role as a source of n u
tritional p r o t e i n . It is t h e r e f o r e a p p r o p r i a t e to investigate n u t r i t i o n a l , p h y siological and u l t r a s t r u c t u r a l f e a t u r e s and to gather as m u c h technological d a ta as p o s s i b l e on large-scale c u l t i v a t i o n of the o r g a n i s m .
The aim of this thesis has b e e n to establish the b a c k g r o u n d of the h e l i c a l structure of Spivulina platensis and the theoretical basis at the root of it.
F u r t h e r m o r e , it w a s considered n e c e s s a r y t o c h a r a c t e r i z e the m o r p h o l o g y and u l tras tructure in relation to environmental f a c t o r s . The environmental factors studied were chosen on the b a s i s of the type of m i c r o - o r g a n i s m and its natural h a b i t a t . C y a n o b a c t e r i a are w e l l k n o w n for their ability to withstand and to g r o w at extremes of t e m p e r a t u r e s . Together w i t h g r e e n algae and d i a t o m s , they are p r o m i n e n t in t e r r e s t r i a l h a b i t a t s in the Arctic and A n t a r c t i c , and many are k n o w n to survive in h o t s p r i n g s at t e m p e r a t u r e s a p p r o a c h i n g b o i l i n g point.These p r o p e r t i e s make a study on the e f f e c t of t e m p e r a t u r e on the m o r p h o l o g y and u l -trastructure seem p r o m i s i n g . T h e effect of light intensity w a s studied because c y a n o b a c t e r i a are p h o t o t r o p h i c m i c r o - o r g a n i s m s . T h e e f f e c t of a v a i l a b l e nitrate is of i n t e r e s t b e c a u s e this a n i o n is n o t u s u a l l y p r e s e n t in rocks of v o l c a n i c o r i g i n , the natural h a b i t a t of Spivulina platensis being volcanic lakes; never
theless , n i t r a t e is t h e b e s t nitrogen source in c u l t i v a t i n g this cyanobacterium (Zarrouk, 1 9 6 6 ) .
R E F E R E N C E S
A i b a , S. and O g a w a , T. 1 9 7 7 . A s s e s s m e n t of g r o w t h yield of a b l u e - g r e e n a l g a ,
Spivulina platensis, in axenic and continuous c u l t u r e . — J. gen. Microbiol.
1 0 2 : 1 7 9 - 1 8 2 .
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B a r o n , C. 1 9 7 5 . Étude d e la d e c o l o r a t i o n des Spivulines. — A n n . N u t r . Alim.
13
29: 6 1 5 - 6 2 2 .
B i s w a s , K. 1 9 2 7 . Aquatic v e g e t a t i o n in B e n g a l in relation to supply of oxygen to the w a t e r . — J. Dep. S c . Calcutta Univ. 8: 49-56.
B o r i e s , G. and T u l l i e z , J. 1975. D e t e r m i n a t i o n du 3,4-benzopyrène dans les
algues Spirulines produites et traitées suivant différents procédés. — Ann.
Nutr. Alim. 2 9 : 5 7 3 - 5 7 6 .
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de p r o v e n a n c e s M e x i c a i n e . — A n n . Nutr. Alim. 2 9 : 5 7 7 - 5 8 8 .
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-trophe, la Spiruline , dans un milieu supplements par 1'acetate de sodium ou
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Am. M i c r o s c o p . S o c . 4 6 : 1 3 9 - 1 4 8 .
Dangeard, P. 1940. A blue alga eaten by m a n : Arthrospira platensis (Nordst.)
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Acad. A r t s S c i . 3 9 : 2 1 3 - 3 2 8 .
Delpeuch, F., J o s e p h , A. and C a v e l i e r , C. 1975. Consummation alimentaire et apport n u t r i t i o n n e l des algues b l e u e s {Osoillatoria platensis) chez quelques
populations du Kanem ( T c h a d ) . — A n n . N u t r . A l i m . 29: 4 9 7 - 5 1 6 .
Farrar, W.V. 1966. T e c u i t l a t l ; a glimpse of Aztec food technology. — Nature 211: 3 4 1 - 3 4 2 .
Feldheim, W., P a y e r , H.D., S a o v a k o n t h a , S. and P o n g p a e w , P. 1973. The uric acid level in human p l a s m a during a nutrition test with m i c r o a l g a e in Thailand. — S.E.A.J. T r o p . Med. P u b . H l t h . 4: 4 1 3 - 4 1 6 .
F i g i n i , G.P. 1925. O s s e r v a z i o n i intorno al genere Spivulina Turp. — Nuov. N d
-tarisia 3 1 - 4 9 .
Ford, T.E. 1980. Life in the p r e c a m b r i a n . — Nature 285: 193-194.
G e i t l e r , L. 1932. Cyanophyoeae. In: Rabenhorst's Kryptogamenflora von
Deutsch-land, Osterreich und der S c h w e i z , vol 14 (Kolkwitz, R. Ed) Akad. Verlag Leipzig.
G e i t l e r , L. 1979. Einige k r i t i s c h e Bemerkungen zu neuen zusammenfassenden D a r -stellungen der M o r p h o l o g i e und Systematik der C y a n o p h y c e e n . — P i . S y s t . E v o l . 132: 1 5 3 - 1 6 0 .
G h o s e , S.L. 1923. A systematic and ecological account of collections of b l u e --green algae from L a h o r e and Simla. — J. L i n n . S o c . London B o t . 46: 3 3 3 - 3 4 6 . G i b b o n s , N . E . and M u r r a y , R . G . E . 1978. Validation of Cyanobaoteviales Stanier
in Gibbons and Murray 1973 as a new Order of the Kingdom Pvoaai-yotae Murray
1968, and of the u s e of neuter plural endings for Photobactevia and Scoto-baetevia classes nov. Gibbons and Murray 1978. — Int. J. Syst. Bacteriol.
2 8 : 3 3 2 - 3 3 3 .
Golubic, S. 1979. C y a n o b a c t e r i a (blue-green a l g a e ) u n d e r the bacteriological code? An ecological o b j e c t i o n . — T a x o n 2 8 : 3 8 7 - 3 8 9 .
Gorbunova, N . p . 1 9 5 8 . T s i t o l o g i y a i sistematicheskoe p o l o z h e n i e Spivulina major
K ü t z . — B o t . Zh. 4 3 : 1 5 8 9 - 1 5 9 3 .
Hedenskog, G., E n e b o , L., V e n d l o v a , J. and P r o k e s , B . 1969. Investigation of some m e t h o d s for increasing the digestibility in v i t r o of m i c r o a l g a e . — B i o t e c h n . B i o e n g . 11: 3 7 - 5 1 .
M a n d e 1 , M. 1979a. Deoxyribonucleic Acid Ease composition of c y a n o b a c t e r i a . — J. g e n . M i c r o b i o l . 111: 6 3 - 7 1 .
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H o l m g r e n , P.R., BOStetter, H.P. and S c h o l e s , V . E . 1971 . ULtrastructurat o b s e r vation of cross-walls In the b l u e - g r e e n alga Sp%TUlvna major. — j . phycol.
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H o l s i n g e r , E.C. 1955. T h e p l a n k t o n algae of three C e y l o n l a k e s . — H y d r o b i o l o -gia 7: 8-24.
I l t i s , A. 1970. P h y t o p l a n c t o n des eaux n a t r o n é e s du Kanem (Tschad). IV Note sur les espèces du genre Oscillatovio sous-genre Spirulina (Cyanophyta) Cahier
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V o p . K r u g o v o r o t a V e s h c h e s t v . Zamknutoi S i s t . O s n . Zhiznedeyatel Nizshikh Organiznov 7: 5 5 - 5 7 . (F.A.A. T r a n s l a t i o n (N.S.) n o . 7 2 ) .
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K e n y o n , C.N., R i p p k a , R. and S t a n i e r , R.Y. 1 9 7 2 . Fatty acid composition and p h y s i o l o g i c a l properties of some filamentous b l u e - g r e e n a l g a e . — A r c h . M i k r o b i o l . 8 3 : 216-236.
K i r c h n e r , O. 1878. A l g e n , p . 1 - 2 8 4 , In K r y p t o g a m e n f l o r a von S c h l e i s e n , v o l . 2 (Cohn, F. e d . ) .
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6 5 - 7 0 .
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R h a n d a w a , M . S . 1936. Occurrence and d i s t r i b u t i o n of the freshwater algae of N o r t h I n d i a . — P r o c . Ind. A c a d . Sci- B. 4: 3 6 - 4 4 .
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15
Generic a s s i g n m e n t s , strain histories and properties of pure cultures of c y a n o b a c t e r i a . — J. gen. M i c r o b i o l . Ill: 1-61 .
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Seshadri , C V . and T h o m a s , S. 1978. Preliminary studies on cultivation of
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Stitzenberger, E. 1 3 5 2 . Spi.TU.Vtna und Arthrospira (nov. gen.) Hedwigia i: 32-34.
Stitzenberger, E. I860. D r . L. Rabenhorst's Algen S a c h s e n s , O b e r - L a u s i t z , Thüringens und N o r d b o h m e n s , systematisch geordnet m i t Zugrundung Eines Neuen S y s t e m s . D r e s d e n .
Turpin, P.J.F . 18 27 . Spinilina Osoillarioide. In: Dictionnaire des Sciences
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facteurs p h y s i q u e s et chimiques sur la croissance et la photosynthese de
1 7 I N T R O D U C T I O N T O I I A N D I I I
The present study was started with an investigation of the cell wall of the c y -anobacterium Spimtlina platensis. The helical morphology as observed under the
light microscope is u n u s u a l as few species possess a helix at the cellular level. Molecular h e l i c e s , on the other hand, are more c o m m o n . T h e DNA double-helix is an outstanding example but many proteins and nucleoproteins are also arranged in this manner. These m o l e c u l e s owe their structure to hydrogen bonds and/or hydrophobic-hydrophilic i n t e r a c t i o n s . Suborganismal helices were recognized by Roelofsen (1950) in the primary cell walls of Phycomyoes sporangiospores, and
their helical growth p a t t e r n s were described by Gamov ( 1 9 7 9 ) . Helical a r r a n g e ments of cellulose m i c r o f i b r i l s are known to exist in many cells of higher p l a n t s , for example in cambial initials, conifer t r a c h e i d s , v e s s e l s , phloem f i bres and sisal f i b r e s , as w e l l as in a number of algae (see Middlebrook and Preston, 1952) . In i n v e r t e b r a t e s , especially in the cylindrical cuticles of some p s e u d o - and eucoelomate w o r m s , the strata fibres are often disposed in c o n centric helices of increasing diameter (Swanson, 1 9 7 4 ) . T h e latter examples are similar in that the h e l i x is linked to the cell w a l l . As for the origin of these helices we may d i s c r i m i n a t e between cellulose or noncellulose bound f o r m s . O r
ganismal helices are a l s o cell wallbound. The best known examples are the S p i -r i l l a c e a e , S p i -r o c h a e t a l e s and Spi-roplasmas (Townsend et a l . , 1 9 8 0 ) , but ce-rtain mutants of BaailluB spp. (Mendelson, 1976; Tilby, 1977 and Fein, 1980) and Seli-beria-llXe micro-organisms (Schmidt and Swafford, 1979) also occur as helices.
The actual helical a r r a n g e m e n t of w a l l components in these bacteria is not a p r e requisite; condition for helical growth and, if p r e s e n t , d o e s not necessarily lead to a helical cell s h a p e . The only factor required is a h e l i c a l or rotational component in c e l l g r o w t h . Tilby (1977) favours the p o s s i b i l i t y of opposed h e l i ces of w a l l p o l y m e r s with unequal stress in them to explain the tighter h e l i c a l growth which appears to reverse in direction w i t h i n the same chain of c e l l s .
There are among the cyanobacteria a few examples of helically arranged t r i
-chomes, such as certain
Lyngbya spp., Anabaena heliooidea, Anabaeyia
spiroid.es
,
certain
Anabaenopsis spp., Osoillatoria ovnata, Osoillatof"ia beggiatoiforirtis,
0 soil lat ort-a boryana, Phovmidium antavoticum and all Spiiv^lina and Arthrospiya
spp. (Pascher, 1925) (see also Booker and W a l s b y , 1 9 7 9 ) . For a better u n d e r standing of the significance of the helix it seems appropriate to deal with itsli
geometry in some details.
A helix is the curve cutting the generators of a right circular cylinder un
der a constant angle 8,
(-•' ~ r cos 9, y = r sin 8, z = r 9 cot B) . Historically
J
o o o
Jthe helix is mentioned by Gerninus (ca 70 BC) , but a passage in Proclus (ca AD
460) suggests that it was already known to Apollonius (ca 225 BC). it was used
by Pappus of Alexandria (ca AD 300) for producing the quadratrix of Hippias of
Elis (a curve r sin 9= (2a/ir)ö, that may be used for dividing an angle into any
number of equal parts). The orthogonal projection of the helix on a plane par
allel to the axis of the cylinder is a curve
as shown in Fig. a. In the literature often
the word spiral is used, where helix is
meant; however, a spiral (of Archimedes)
(e.g. r = a6 + b) is a two-dimensional curve
which was discussed first by Archimedes
about 225 BC.
The main characteristic of a helix is its
pitch, P (2 7T r /cot B) , which corresponds to
the axial distance along the helix that
gives a rotation of 2 T (360 ) on its surface.
Another characteristic is the screw-angle of
the helix I, the angular displacement of
successive mathematical structural units. It
is now evident that:
L = 2 TT p / P
where p is the separation between units. (As
will be evident from Fig. a,
1. is the pro
jection on the z,0-plane of the angle be
tween the axis of two successive units.) In
biomolecules these subunits may be
nucleo-tides as in DNA. When viewed under the elec
tron microscope, the image normally gives a side-on view (elevation) of a helix
with an axial (z) repeat P and a radius r (see Klug, Crick and Wyckoff, 1958).
o
A helix is usually defined in terms of the cylindrical polar coordinates r, $
and z (see Fig. b ) . The density variations in the helix satisfy the relation:
Fig. a. Side-on view (elevation)
of a helix with an axial
(z) repeat P and a sepa
ration p between struc
tural subunits.
p (r,4-,z) = p (r,<f>,z + P) ,
where P is the repeat distance of the helix in the z-direction.
Biological helices are not continuous distributions of density but are a
19
series of identical subunits (in this c a s e , eel Is) separated in the z-direction by a d i s t a n c e , p , on a helix of pitch P as shown in Figure b. These subunits are successively rotated through the screw-angle t.
Initially, it will be assumed that there is an exact number of subunits in a distance P, that is P/p is an integer, each subunit being rotated by Y_ = 2 ;i p/P with respect to the
one below or above it.
Fig. b. 2-projection of a helix in p o l a r The discontinuous helix can be c o o r d i n a t e s . . , , . _
represented as the multiplication or
a continuous helix by a one-dimensional lattice with a repeat distance p (Sher
wood , 1976; see also M i s e l l , 1 9 7 8 ) . Mendelson (1976) defines the helix angle of sarface o r g a n i z a t i o n in Bacillus spp. as a tangent helix angle = pitch/circum
ference, where circumference equals D (D diameter of the h e l i x ) . /mother c h a -2 -2 racteristic is the helix length defined by (helix length) = (pitch) (circum-ference) . Mendelson (1976) states that for h e l i c a l growth m o r p h o l o g y , it is necessary to assume that new cell surface is inserted along a helical path and thus the major w a l l components (for cyanobacteria peptidoglycan) must in some respect fit in this o r i e n t a t i o n . One simple possibility is that the glycan backbone of the p e p t i d o g l y c a n might be oriented in a helical p a t h . This would provide a structurally sound organization for the cell w a l l .
Spintl-ina and Arthrospira spp. are the only cyanobacteria to have a well d e
veloped helical shape w h i c h is a constant p r o p e r t y , and therefore the most s u i table organisms for the study of the organismal biological h e l i x . In this study
Spt-rulina platensi-SVas chosen for obvious reasons. Chapter II represents an
integral study of the cell w a l l proper and the c r o s s - w a l l . The latter did not fit previously described morphological m o d e l s for cell wall shape. During the cell wall studies a fibrillar layer b e t w e e n the plasma m e m b r a n e and the p e p t i doglycan layer was d i s c o v e r e d . An additional study using pyrolysis m a s s s p e c -trometry was initiated to elucidate the chemical nature of these f i b r i l s . This is described in chapter V. The in vitro shape of the cross-wall as found in II resulted in several h y p o t h e s e s regarding the origin of its m o r p h o l o g y . T h e s e are described and analyzed in chapter I I I .
20
REFERENCES
Booker, M.J. and Walsby, A.E. 1979. The relative form resistance of straight and helical blue-green algal filaments. — Br. Phycol. J. 14: 141-150. Fein, J.E. 1980. Helical growth and macrofiber formation of Bacillus subtilis
168 autolytic enzyme deficient mutants. — Can. J. Microbiol. 26: .330-337. Gamov, I. 1979. origins of spiral growth, — New Scientist 83: 652-654. Klug, A., Crick, F.H.C. and Wyckoff, H.W. 1958. Diffraction by helical struc
tures. — Acta Cryst. 11: 199-213.
Mendelson, N.H. 1976. Helical growth of Bacillus subtilis: A new model of cell growth. — Proc. Natl. Acad. Sci. USA 73: 1740-1744.
Middlebrook, M.J. and Preston, R.D. 1952. Spiral growth and spiral structure. — Biochim. Biophys. Acta 9: 32-48.
Misell, D.L. 1978. image analysis, enhancement and interpretation ch. 3 (In: Practical methods in electron-microscopy vol. 7 A.M. Glauert ed. ) North Holland publishing Cy., Amsterdam.
Pascher, A. 192 5 . Süsswasser-flora Deutschlands, Osterrei chs und der Schweis. Heft 12: Cyanophyceae (bearbeitet von L. Geitler). G. Fischer Verlag, Jena, Roe lof sen, P -A. 1950. The origin of spiral growth in Phy corny ces sporangio spores.
— Ree. Trav. Bot. Néerl. 17: 73-110.
Schmidt, J.M. and Swafford, J.R. 1979. Isolation and morphology of helically sculptured, rosette-forming, freshwater bacteria resembling Selibevia. — Curr. Microbiol. 3: 65-70.
Sherwood, D. 1976. Crystals, X-rays and proteins. — Longman Group, London. Swanson, C.J. 1974. Application of thin shell theory to helically wound fibrous
cuticles. — J. Theor. Biol. 43: 293-304.
Tilby, M.J. 1977. Helical shape and wall synthesis in a bacterium. — Mature 266: 450-452.
Townsend, R., Burgess, J. and Plaskitt, K.A. 1980. Morphology and ultrastruc-ture of helical and nonhelical strains of Spiroplasma citri. — J. Bacteriol. 142: 973-981.
21
Antonie van Leeuwenhoek 43 (1977) 89 99
I I . On the morphology and ultrastructure of the cell wall of
Spirulina platensis
C. VAN EYKELENBURG
Laboratory of Microbiology, Delft University of Technology,
Delft, The Netherlands
van EYKELENBURG, C. 1977. On the morphology and ultrastructure of the cell
wall of Spirulina platensis. Antonie van Leeuwenhoek 43: 89-99.
The cell wall of the cyanobacterium Spirulina platensis was studied with the
electron microscope using ultra-thin sectioning, shadowing, carbon-replication
or freeze-etching techniques for specimen preparation. The cell wall could be
resolved into four layers, L-I through L-IV. The L-I and L-III layers contain
fibrillar material. The septum is a three-layered wall: an L-II layer sandwiched
between L-I layers. The shape in vitro of isolated septa might be an artifact due
to the preparation technique used. Certain structural properties of the septum
seem to allow tangential stretching; they might be reflected in the flexible gliding
mobility of Spirulina species. The outer, L-IV layer contains material longi
tudinally arranged along the trichome axis.
INTRODUCTION
some cyanobacteria and green algae are considered to be potential sources of
nutritional protein (Soeder, 1976). One of the most promising species is
Spirulina platensis because of the technological advantages it
offers (Soeder, 1976).
A review article by Wolk (1973) gives integrated information on
the ultrastructure of the cyanobacteria. Since little is known about the
morphology and ultrastructure of Spirulina species an electron microscopic
study was initiated.
The cell wall of cyanobacteria consists of four layers, L-I through L-IV
(Jost, 1965) though Jensen and Sicko (1972) reported an eight-layered wall
for Gloeocapsa alpieola.
Allen (1968) suggested the L-I layer to be an artifact of preparation. The
L-II layer contains peptidoglycan as has been repeatedly found in various
species and which is generally accepted for cyanobacteria (Halten, I973).
In this L-II layer rows of pores are frequently visible on both sides of the loci
22
C. VAN ËYKELENBURG
of ingrowth of the septa m several filamentous cyanobactena (Metzner,
195.V Halfen and Castenholz, !
L'71). Metzner( 1955) suggested that these pores
are involved in mucilage secretion. The L-111 layer of Oscillatoria princeps
was shown to be libnllar and proteinaceous (Llalfen. 1973) To oar knowledge
very little is known about the infrastructure of the L-IV layer.
The present article deals with the morphology and ultrastructure of the cell
wall and the septum o\~ Spirit!ina platensis. The results were compared with
literature data on other cyanobactena. Special attention was paid to the
shape of the septum, which was considered in relation to the helical shape
of the trichome.
MATERIALS AND METHODS
Culture methods. An axenic strain of Spirulinaplatensis [from Lake Nakuru,
Africa] and a
\enie
stram of Spirulina laxissima (from Lake Nakuru. Africa)
were cultivated in a medium according to Ogawa and Terui (1970) using tap
water instead of demineralized water. The organisms were grown in 100 ml
Erlenmeyer flasks at 25 C The light intensity of the fluorescent lamps by which
the cultures were illuminated was 5 klux The trichomes were harvested alter
10 to 18 days
Isolation of cell walls. Cells were disintegrated in a Vliekle shaker | Hampton
Middx, UK., 50 Cycles! with ballotmi beads, 0.17 0.18 mm in diameter at 4C
for ten minutes in the presence of 0.001",, (w v) DNAse (from beet pancreas:
NB cy). The suspension was made up in a 0.01 M EDTA solution. Cell walls
were separated from plasma and cellular organelles by centrifugation at 2000 g
at 4C and washed twice with demineralized water. A fraction resistant to
sodium dodecyl sulphate (SDS) was isolated by treatment o\' the isolated cell
walls with a hot (100C) 4",, SDS solution for a few seconds.
Preparation for sectioning. Cell walls were fixed in 2.5",, glutaraldehyde in
a 0.2 M phosphate buffer (pH =7.4) according to Hayat (1
LJ72) with 0.2",,
sucrose for 2 hours at 20 C. The material was washed twice in buffer followed by®
embedding in Spurr and ultra-thin sectioning followed. Sections were stained
with lead citrate and uranyi acetate.
Preparation for shadowing. Cell walls and the SDS-resislant fraction were
washed in demineralized water and platinum-shadowed at 10
:iTorr
Preparation for replica technique. Replication was carried out with untreated
material by the platinum/carbon replica technique. A 3.5",, potassium
dichro-mate solution in 25",, sulphuric acid and a saturated sodium hypochlorite solu
tion were used to clean the replicas. Alter washing the replicas with distilled
water they were examined electron microscopically.
Preparation for freeze-etching. A 20"
odimethyl sulfoxide (DMSO) solution
in culture medium was used as a eryoprotectant. The cells were quickly frozen
® post_fixation in ! % O s 04 for 2 h o u r s a t 2 0 C after which
23
G 11 WALL or SPIRULINA PLATENSIS
after a 20-minute treatment wuh DMSO Etching was done at 105 C at 5.10""
I orr and lasted 2 minutes The freeze-etch replicas were cleaned as described
before.
Experiments with pepsin (Merck. 10000 E g. 71 85), trypsin (Miles-Seravac,
bovine), lysozyme (ELBG, 15351) and pronase (Calbiochem, 45000 PUK/g,
53 702) were carried out according to Braun and Rehn 11969).
RES Li LTS
Electron microscopy of ultra-thin sectioned material of Spirulina platensis
revealed the four-layered longitudinal cell wall (Fig. I). Fig. 1 also shows the
ingrowth of a septum in statu nascendi, which is three-layered: an L-II layer
1
L| Lll Llll LIV
—T-_
.
-ST" /
W&r&'a
Fig. i. Section through S. platensis. The cell wall is divided into four layers, the septum i
divided into three layers.
The black bar indicates 500 nm unless otherwise stated.
«ss
III::
m
- pepiiduglvc; L-l - f i b r i l s
24
C. V A N E Y K E L E N B U R G
sandwiched between L-I layers. L-I and L-III are electron transparent whereas
the L-II and L-IV layers are electron-dense. All layers are 10 to 15 nm thick and
therefore the whole wall ranges in thickness up to about 60 nm. The L-II layer
in the septum and in the longitudinal cell wall equal each other in thickness
but the L-I layers seem to have expanded.
Untreated septum material is almost always accompanied by fibrils as seen
in Fig. 2. The diameter of these fibrils is 14 to 15 nm.
In untreated cell walls the L-III layer appears to be fibrillar (Fig, 3). The
fibrils were continuous over the trichome surface in a right-handed helix. Their
diameter was 8 to 10 nm.
Replica experiments revealed the outermost layer (L-IV) of the cell wall
(Fig. 4) to be composed of linearly arranged material. The size of the elements
of this arrangement is 12 to 15 nm and the direction of the array is parallel to
the trichome axis. Distortion of the cell wall leads to a distortion in the normal
Fig. 2. Shadowed septum showing fibrils ascribed lo the L-I layer.
25
CELL WALL OF SPIRULINA PLATENSIS
Fig. 3. Shadowed specimen of the L-III fibrils.
GËÉ
m.
Fig. 4. Replicated trichome of 5. platensis with linearly arranged material from the L-IV layer. The arrow indicates a septum.
arrangement (Fig. 5). Small areas are found in which the former arrangement
is still present though these areas themselves form a distorted pattern.
Isolated septa visualized by shadowing are round and characteristically
shaped (Fig. 6). They appear as thin discs which are folded in a sector covering
about 5% of the total septum surface (Figs. 6 and 7). This implies that there is
10% more septum material than necessary for a flat septum.
2 6
C . VAN EYKELENBURG
5^ M
^T*
■■■■"■'-^.. i
Fig. 5. Distorted cell wall with a distorted pattern of the L-IV layer.
Fig. 6. Shadowed specimen of a septum with a fold and a zipper.
27
CELL WALL OF SPIRULINA PLATENSIS
fig. 7. Shadowed septum-part clearly showing the fold. The dark line represents 100 nm.
?&»
^;'
^L
' S
v, .,»,,>,
28
C. VAN EYKELENBURG
but with a larger pitch was used to find a correlation between the shape of the
septum and the trichomc morphology. Fig. 8 shows a septum of Spirulina
laxissima which is folded in a similar way while the sector covered is about 3%
of the total surface.
The purpose of the freeze-etch experiments was to create a fracture face
through all cell-wall layers to visualize the respective ultrastructural features.
The fracture faces obtained were situated in the L-Il layer, in the plasmalemma,
or in the cytoplasma. The L-II layer could be recognized by the rows of pores
near the septa.
Enzymes and chemicals were used in efforts to create another weak place in
the cell wall leading to another fracture face. The experiments carried oui with
enzymes such as pepsine, trypsine, pronase and lysozyme, and with chemicals
such as SDS and sodium hypochlorite in different concentrations failed to give
the desired result. However, some results should be reported. Figs. 9 and 10
show a zipper-like structure in the L-II layer at a septum crossing. In Fig.6 the
zipper itself is wholly isolated from the longitudinal cell-wall but still half
Figs. 9 and 10. Freeze-etched zipper structures.
CELL WALL OF SPIRULINA PLATENSIS
11
^/w&itöiA- ■ . .■■jmw&as-■
Fig. II. Visualization of the rows of pores in ultra-thin sectioned materia
*1/Z;
Fig. 12 Inner fracture face of the L-II layer showing the membrane-associated particles.
attached to a septum. In ultra-thin sectioned material rows of pores could be
seen in the same place as shown in Fig. 11.
Another phenomenon to be reported is the characteristic pattern of mem
brane-associated particles in the L-II layer. Fig. 12 shows the distribution of
these particles.
30
C. VAN EYKELENBURG
DISCUSSION
The four-layer structure of the longitudinal cell-wall and the three-layer
structure of the septum are consistent with observations by Jost (1965) and by
Halfen and Castenholz (1971) on Oscillatoria species.
The fibrils of Fig. 2 are assumed to originate from the L-I layer although
they were never seen as a layer on the septum. This phenomenon could he due
to the presence of a matrix in which these fibrils might be embedded, whilst
the attachment of the L-I layer to the rest of the cell wall could be very loose.
The assignment of the fibrils to the L-I layer is based on ruling out other possi
bilities. Experiments have shown that these fibrils are resistent to sodium
dodecyl sulphate concentrations up to 4%.
Halfen and Castenholz (1971) and Halfen (1973) discussed an L-III fibrillar
layer and its function in the gliding movement of Oscillatoria princeps. The
diameter of the L-III fibrils of this organism was 5 nm. Halfen (1973) found these
fibrils to be proteinaceous and wound helically around the tnchome. The same
conclusion applies to Spirulina platensis. The regular pattern in the L-IV layer
was sofar unknown in cyanobacteria but resembles the ultrastructure of the
outermost cell-wall layer of gram-negative bacteria described by Thornley.
Glauert and Sleytr (1974). The distortion of the structure in the L-IV layer of
Spirulina platensis is probably due to the construction of the layer and to the
material of which it is composed.
The morphology of the septum in vitro might implicate a different morpholo
gy in vivo. However, it might merely be an artifact, due to the preparation
technique used. Theoretical consideration of the exact shape of the septum in
vitro suggests that it might result from tangential stretching of either one of the
two layers in the septum or both of them during preparation. Certain structural
properties of these layers common to both the cell wall proper and the septum
might be responsible for the flexible gliding motion of Spirulina species on the
one hand and the stretchability of the septum on the other. Work to provide
evidence for this hypothesis is in progress (Van Eykelenburg, Fuchs and
Schmidt, to be published). Remarkably, the size of the area covered by the fold is
related to the pitch of the trichome. The larger the pitch the smaller the
the folded area and vice versa. Spirulina laxissima was used to illustrate the
hypothesis. Fig. 4 supports the correlation. The phenomenon described fits
in the helical macro-morphology of the trichome or vice versa.
Metzner's (1955) idea that the rows of pores on both sides of the septa in the
longitudinal cell-wall may play a role in mucilage secretion must be reconsid
ered. Figs. 6, 9, 10 and 11 suggest that the zipper-like structure is rather loosely
built into the trichome and therefore could represent a trichome propagation
device. The propagation mechanism might result in a tearing apart right
through the L-II layer of the septum. Another mechanism could be that the
septum as a whole together with the zipper is removed so that two cells would
31
CELL WALL OF SPIRULINA PLATENSIS
be killed. The latter mechanism is supported by Fig. 6.
In general the cell wall of Spirulina platensis can bv
schematically represented as in Scheme 1.
Chemical characterization of the different lypes of cell-wall layers will be the
next step in comparing Spirulina species with other cyanobacteria.
1 wish to thank Ir B. H. A. van Kleeff, Dr Ir P. Kooiman and Drs P. J.
Nieuwdorp for their kind support and Prof. Dr A. Fuchs and Prof. Dr
T. O. Wikén for critically reading the manuscript.
Received 2% February 1977
R E F E R E N C E S
ALLEN. M M. 1968. Ultrastructure of the cell wall and cell division of unicellular blue-green algae. - J . Bacteriol. 96: 842-S52.
BRAUN, V. and REELS. K. 1969 Chemical characterization, spatial distribution and function of a lipoprolcm (murem-lipoprotcin) ol'lhc E eoli cell wall. Eur J. Biochcm 10:426 438.
HALFEN, L. N. and CASTENHOLZ. R W . 1971. Gliding motility in the blue-green alga Oscilla toria princeps - J . Phycol. 7 : 133 145.
HALFEN, L. N 1973 Gliding motility of Oscillatoria Ultrastructure and chemical characteri zation of the fibrillar layer.-- J. Phycol 9: 248 253.
HAYAT. M. A. 1972. Basic electron microscopy techniques, p. 97.—Van Nostrand Reinhold Company. New York.
JENSEN. T. E. and SlCKO, L. \ . 1972. The fine structure of the cell wall ofGloeocapsa alpicola, a blue-green alga. —Cylobiologie 6: 439-446
JOST, M. 1965. Die Ulirastruktur von Oscillatoria rubescens D C -Arch Mikrobiol 50: 211-245.
METZNER, I. 1955. Zur Chemie und zum submikroskopischen Aufbau der Zellwande, Scheiden und Gallenen von Cyanophyceen.— Arch. Mikrobiol. 22:45-77.
OGAWA, T. and TERLI, G. 1970. Siudics on the growth of Spirulina platensis. I. On ihe pure culture of Spirulina platensis —J. Ferment. Technol. 48: 361- 367
SOEDER, C. J. 1976. Zur Venvendung von Mikroalgcn fiir Ernahrungszwecke. Naturwis-sen.schal'ten 63: 131 138
THORNLEY, M. J.. GLAUERT. A. M and SEEYTR, U. B. 1974. Structure and assembly of bacterial surface layers composed of regular arrays of subunits.—Phi] Trans R Soc Lond. B 268:147-153
WOLK, C, P. 1973. Physiology and cytological chemistry of blue-green algae.- Bacteriol. Rev. 37: 32-101.
33
J. theor. Biol. ( 1 9 8 0 ) 8 2 , 2 7 1 - 2 8 2
I I I . Some Theoretical Considerations on the In Vitro Shape
of the Cross-walls in Spirulina spp.
C. V A N E Y K E L E N B U R G
Laboratory of Microbiology
A . F U C H S
