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Biol Fertil Soils 1986) 2:131-146
Biology and Fertility
of S o f t s
© Springer-Verlag 1986
Chemical composition of cultures and natural samples
of Nrfixing blue green algae from rice fields
P.A. Roger , A. Tirol, S. Ardales, and I. Watanabe
The Int ernat iona l Rice Research Institute Los Bfinos, Laguna, Philippines
Summary.
Laboratory cultures, soi l cultures, and
natural samples of N2-fixing blue-green algae (B GA )
from rice fields were analyzed for dry matter, ash, N,
C, P, and a few other consti tuents.
Results show a very large variabil ity of the composi-
t ion. Dry ma tter contents ranged from 0.28 to
13.6 (average 3.3 ). Ash contents ranged from
15.6 to 71.3 . Nitrogen contents ranged from
1.9 to 11.8 on an ash-f ree basis (aver age 6 ).
Carb on conte nt was less variable, ranging from 37
to 72 and averaging 43.7 .
A decrease in N and pigment contents, and an
increase in reducing sugars, was observed in aging
laboratory cultures.
Large differences in composit ion were observed
betw een field samples and material grown in artificial
medium. Soil-grown BGA and field samples were
character ized by very high ash contents, N con tents
lower than those in laboratory cultures, and P defi-
ciency.
Extrapolat ion from (1) average dry matter, ash,
and N contents and (2) records of BGA biomass in
rice fields indicates that an algal bloo m has a poten tia-
l i ty of about 15-25 kg N per hectare and that a BG A
biomass of agronomic significance is visible to the
naked eye .
Key words:
Blue-green algae - Chemical composi-
tion
N 2
fixat ion - Rice fields - Cyanob acteria
Offprint requests to P.A. Roger , IRRI, PO Box 933, Manila,
Philippines
* Visting Scientist, ORST OM, France
Despite the abundance of l i terature on the role of
N2-fixing blue-green algae (B GA ) and on the ir pos-
sible use as a sou rce of ni trogen for rice (Roge r and
Kula soor iya 1980), little is know n about thei r compo -
si t ion. In book s and reviews on BG A, discussions on
overal l composit ion, i f any, are often very short and
the av erage values ei ther are calculated from limited
data (Fogg et al. 1973; Wo lk 1973) or are no t suppo rt-
ed by bibliographic refe rences (Mishustin and Shil '-
nikova 1971).
A comprehensive knowledge of the composi tion of
BG A would be necessary for the proper evaluat ion of
their agronom ic significance.
Frag men tary information is available m ainly from
physiological studies with laboratory strains grown
under art ificial condit ions. Quanti tat ive data on N,
protein, C, carbohydrates, P, and ash contents in
Nfix ing BGA, co l lec ted from the l i te ra ture , a re
pres ented in Table 1. Nitrogen content averages 7
and exhibits a fourfo ld range of variat ion (2.8 to
11 ). Carbon conten t is less variable (38 -48 ).
Carbon: ni trogen rat ios range from 4.3 to 7.4. Aver-
age values, calculated from a restricted number of
data obtained with 14 species in 6 genera, a re hardly
representat ive and are presented only to sat isfy the
reade r's intel lectual curiosi ty.
This paper summarizes analyses of Nfix ing B GA:
(1) grow n und er art ificial condit ions in the laborato ry
as flask or mass cultures in l iquid med ium, (2) grown
on soil in a greenh ouse, and (3) collected from
natura l envi ronments .
Results of a study on the mineral izat ion of some
BGA in soil are also presented. Potential i ty and
implicat ions for ut i l izat ion of BGA as a N source for
rice are discussed.
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132
T ab le 1 . Da ta on co mpo s i t i on o f N2 - f i x ing BGA c o l l ec t ed f r om the l i t e r a tu r e
P . A. Rog er e t al . : Compos i t i on o f b lue - g r een a lgae
R e f e r e n c e S p e c ie s R e m a r k s N C a r b o h y - C C : N P A s h
d r a t e s
Co l lye r and A n a b a e n a 11 d a y s old 8 .6 32 . . . .
F o g ( 1 9 5 5 )
c y l i n d r i c a
33 d a y s o ld 6 . 9 24 . . . .
C o b b a n d
A . c y l i n d r i c a
10 . 4 - 48 4 . 6 1 . 8 /2 . 1 6 /7
M y er s ( 19 64 ) 2 d a y s o ld 5 . 0 . . . . .
F o w d e n ( 1 9 5 4 ) A . c y l i n d r i c a 5.6 . . . . .
C h o i a n d
A . f l o s a q u a e
O n N 2 , u r e a 10.1 . . . . .
M a r k a k i s ( 1 9 8 1 ) o r p e p t o n e
A . f l o s a q u a e
O n n i t r a t e 8 . 0 . . . . .
A . f l o s a q u a e
O n n i t r i t e 6 . 4 . . . . .
K e C h a n g D a n g a n d
A. variab i l i s
E x p o n e n t i a l 4 . 8 4 3 . . . .
N i k i t i n a ( 1 9 8 0 ) p h a s e
A . v a r i a b i l i s
L ys is p h a s e 3 .2 2 0 . . . .
H e a l e y a n d
A . v a r i a b i l i s
P su f f i c i en t 10 . 5 20 45 4 . 3 1 . 56 -
H e n d z e l ( 1 9 7 5 ) A. variab i l i s P de f i c i en t 9 . 0 43 48 5 . 3 0 . 70 -
A . v a r i a b i l i s
P def ic ient 5 .1 50 38 7.4 0 .13 -
S h n y u k o v a e t
A p h a n i z o m e n o n
Natu r a l b loo m 4 . 8 13 - - 0 . 45 -
a l . ( 1978)
f l o s a q u a e
Natu r a l b loo m 6 . 7 - - - 0 . 63 -
T inda l e t a l . A p h a n o t h e c e O n 1 a n d 2 M NaC1 9 /1 1 . . . . .
( 1 9 7 7 )
h a l o p h i t i c a
O n 3 M N aC 1 1 0 / 6 .9 . . . . .
D e C a n o a n d D e
A . s t a g n i n a
E x p o n e n t i a l 6 . 7 . . . . .
H a lp e r in ( 1 97 8 ) p h a se . . . . .
Wi l l i ams and
C a l o t h r i x
3.7 . . . . .
Bur r i s ( 1952) p a r i e t i n a
K o k y r s t a a n d N o s t o c l i n c k i a 4 0 / 8 5 . . . .
C h e k o i ( 1 9 7 2 )
Wi l l i ams and
N . m u s c o r u m
6 .0 . . . . .
Bur r i s ( 1952)
M e h t a a n d
N o s t o c
sp. - - 4 8 . . . .
V a i d y a ( 1 9 7 8 )
T i r o l e t a l . ( t 983)
N o s t o c
sp. - 7 .3 - 38 5.3 1 .13 -
H a r p e r a n d No d u l a r i a 2 . 8 O . 1 8
Danie l ( 1935)
s p u m i g e a
Aver age 7 . 0 38 43 5 . 4 0 . 96 6 . 5
Coef f i c i en t o f va r i a t i on 34 53 12 22 74 -
aterials and metho ds
C o n d i t i o n s o f g r o w t h
F l a s k c u l t u r e s i n l i q u i d m e d i u m . R e g u l a r c u l t u r e s o f o u r B G A
col l ec t i on ma in t a ined i n 300- ml E r l enmeyer f l a sks con t a in ing 100
m l G O m e d i u m ( R i p p k a e t a l. 1 9 7 9) a n d i n c u b a t e d a t t h e a m b i e n t
t e m p e r a t u r e u n d e r c o n t i n u o u s i l l u m i n a ti o n p r o v id e d b y w h i t e c o l d
f luo r escen t t ubes ( 600 lux ) wer e used . Fo r ty - s ix samples co r r e -
spond ing to 32 s t r a ins o f 10 gener a wer e ana lyzed . T wen ty - two
cu l tu r es wer e co l l ec t ed b e f o r e 4 weeks o f g r owth and the i r d r y
m a t t e r , N , C , r e d u c i n g s u g a r s , c h l o r o p h y l l , a n d p h y c o c y a n i n
c o n t e n t s w e r e m e a s u r e d . T w e n t y - f o u r o t h e r c u l t u r e s w e r e c o l le c t-
ed be t ween 4 and 8 week s o f g r owth and wer e s imi l a r ly ana lyzed .
M a s s c u l t ur e s i n l i q u i d m e d i u m . Seven st rains were grown in 20-1
c a r b o y s , i n G O m e d i u m w i t h N a2 C O 3 c o n c e n t r a t i o n i n c r e a s e d
t en f o ld , con t inuo us bubb l ing wi th 0 . 5 CO~ in a i r , con t inu ous
s t i r r ing , an d l i gh t ing . L igh t i n t ens i t y was i nc r eased a long the
g r owth pe r iod us ing one t o s i x f l uo r escen t t ubes p l aced ve r t i ca ll y
20 cm away f r om the ca r boys . Cu l tu r es wer e ha r ves t e d a t t he end
of t he l oga r i t hmic phase o f g r owth .
A r t i f i c i a l b l o o m s . I n a g r ee nhou se exper im en t i n 0 . 5 - m 2 t r ays ,
d e s i g n e d t o s t u d y t h e g r o w t h o f i n d i g e n o u s o r i n o c u l a te d B G A o n
f our submer ged so i l s ( ac id i c , neu t r a l , a l ka l i ne , and pea t so i l s ) ,
b looms deve loped a t t he f l oodwate r su r f ace . E xper imen ta l cond i -
t i o n s e n s u r e d m i n i m u m d i s t u r b a n c e i n t h e t ra y s . W a t e r w a s m a i n -
t a i n e d a t 5 c m d e p t h w i t h d e m i n e r a l i z e d w a t e r a n d n e v e r b e c a m e
m u d d y . O n e m o n t h a f t e r s u b m e r s i o n o f t h e s o i ls , f lo a t in g b l o o m s
wer e ha r ves t ed , t ak ing ca r e no t t o d i s tu r b t he so i l .
S o i l b a se d i n o c u l a .
Alga l ma te r i a l was p r o duced in sha l low t r ays , 1
m x lm , ma de wi th a po lye thy l ene f i lm p l aced on a wood en f r ame
on t ab l es i n a g r eenh ouse . S ix s t r a ins wer e f i rs t g r own in 20- 1
c a r b o y s o n G O m e d i u m . C u l t u r e s w e r e h a r v e s t e d s h o rt l y a ft e r t h e
logar i t hmic phase o f g r owth and inocu la t ed i n to t he t r ays . One da y
bef o r e i nocu l a t i on each 1 - m2 t r ay r ece ived 5 kg s i eved d r y M aaha s
so i l (aqu i c T r opuda l f , pH 6 . 9 ) , 10 g super p hosp ha t e , 2 g NaC1 , 2 ml
e thy l an ( t o con t r o l g r aze r s ) , and was f l ooded to 5 cm wi th deminer -
a l i zed wa te r . T h i s me tho d o f p r oduc ing so i l - based i nocu lum was
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P.A. Roger e t a l .: Compos i t i on of b lue-green a lgae 133
most eff icient under dry season co ndi t ions in the Phi l ippines ( IRR I
1985). A fter 2 weeks , m ost of the algal mat that develo ped at the
soi l-water i n ter f ace became detached and f loa t ed a t t he f loodwater
surface in t rays inoculated with Anabaena Aulosira Nostoc and
Tolypothrix.
For
Fischerella
a n d
Scytonema
an algal growth was
obviou s at the soi l surface and 0 2 bubb les were vis ible, but the mat
nei ther became detached nor f loa t ed. F loat ing a lgal mat s and the
upper layer of the soi l colonized by algae were col lected and
combined in each t ray. Being highly contaminated with soi ls , this
mater i a l cannot be cons idered only as BGA. Data have been
included because informat ion on the compos i t i on of so i l -based
inocula i s l acking and to provide informat ion on the potent i a l
biomass of natural algal blooms. Nit rogen content in the ash-free
algal mater ial was calculated on th e bas is of a 40% carbon c onten t
i n B G A .
Field samples. Float ing colonies or algal masses were col lected
from r ice f ields in the IRR I farm a nd the surroun ding area us ing a
net , 20 cm in diameter , 1 mm mesh. Mater ial was r insed with r ice
f ield f loodw ater and o rganic debr is sor ted out . An alys is was
performed shor t ly af ter col lect ion.
Strains.
Thir ty s t rains of ten genera have been used for analys is
(Table 2) . S t r a ins obta ined through the cour t esy of Dr . Rippka
(PCC s trains of Pas teur Ins t i tute) were axenic. Ot her s t rains were
unialgal . Two s t rains of Oscillatoria a genus usual ly considered
non-N2-f ixing in aerobios is , hav e also been included.
Methods of analysis. Dry weight and ash were det ermined on
pel lets f rom algal suspension cen tr i fuged at 10 000 rpm for 15 min.
Dry weigh t was measu red af ter 24 h of heat ing at 80°C in an oven,
and ash content by heat ing the mater ial at 325°C unt i l smoking
ceased an d th en a t 480°C overnight . Minera l content s were measur -
ed us ing analyt ical methods for plants of the IRRI analyt ical
l abora tory .
Prote ins , carbon, sugar s , and p igment s were measured us ing
algal suspensions sonicated for 5 min (Sonicator Model W1851,
Heat Systems-Ultrasonics , Inc.) in an ice bath. Protein content
was measured by the Fol in-Ciocal t eau phenol method (Lowry e t
al . 1951) on samples hydrolyzed with NaOH for 1.5 h at 60°C.
Bovine s erum a lbumin was used as s t andard . Carbon was measur -
ed by the Walkley and Black method (Black 1965) . Sugars were
mea sured b y the phenol-sulfur ic acid metho d (Dub ois et al. 1956).
Chlorophyl l a and phycocyanin were measured f rom the absorp-
t ion spectra of sonicated mater ial according to the equat ions of
Myers and Kratz (1955) . Ni t rogen in ar t i f icial BGA blooms was
measured by the s t andard micro-Kjeldahl method. Data , except
dry mat ter , are expressed on a dry weight bas is .
Statistical analysis.
Resul ts of analys is of the 46 samples grown in
f lask cul tures were used for cross-correlat ion s tudy an d clus ter and
mult ivar iate analys is , ut i l izing the computer faci l i t ies and the
CLU STA N program of t he Agr icul tural Research Center of t he
Univer s i ty of t he Phi l ippines a t Los B~nos . Dendrograms were
drawn f rom three major component s us ing Ward ' s method of
pool ing.
Mineralization study. Water - sa tura t ed soi l f rom the IRRI f arm
(Maahas soi l ) was passed through a 2-ram s ieve. A quant i ty
equiv alent to 12.5 g dry weight was placed in a s toppere d glass tube
2 cm in diameter and 25 cm in length. Algal and control mater ials
equiva lent to 3 mg N (120 ppm dry soi l) were placed on th e soi l and
covered with the same quant i ty of water-saturated soi l . Dis t i l led
water was add ed to a height of 4 cm above the soi l surface. Tubes
Table 2. Strains and number of samples ut i l ized in the dif ferent s tudies
Strain Origin A B C D Ref . No.
Anabaena variabilis
UPLB, Phi l ippines , Dr . Mart inez 1 1
Anabaena sp. C.A. Dr . Van Baalen 1 1 2
Anabaena
sp. Dr . New ton 1 3
Anabaena sp. China 1 4
Anabaena
sp. PCC 7120, Dr . RiPpka 1 1 5
Anabaena
sp. PCC 7122, Dr . Rippk a 1 6
Anabaena sp . IRRI Greenhouse 1 7
Aphanothece
sp. IRR I farm, Uppe r MN 1 8
Aulosira fertilissima Cambridg e cul ture col lect ion 1 1 1 9
Calothrix sp. PCC 7101 , Dr . Rippk a 1 10
Calothrix spp. (2) Uppe r Banau e, Phi l ippines 1 11 - 12
Calothrix spp. (3) Banaue, Phi l ippines 1 6 13 -15
Fischerella
spp. (2) Kiangan, Phi l ippines 2 16 -17
Fischerella sp. Banaue, Phi l ippines 1 1 1 1 18
Gloeotrichia sp. UPLB, Phi l ippines , Dr . Mart inez 1 19
Gloeotrichia
spp. (2) Laguna, Phi l ippines 1 4 20 -21
Nostoc
sp. Sr i Lanka, Dr . S.A. Kulasoor iya 1 3 1 22
Nostoc spp. (2) UPLB, Phi l ippines , Dr . Mart inez 2 2 23 -2 4
Nostoc
sp. PCC 7310 2, Dr . Rippk a 1 25
Nostoc
sp. Uppe r Banaue, Phi l ippines 1 3 26
Nostoc sp. Banaue, Phi l ippines 1 27
Nostoc sp. Luis iana, Phi l ippines 1 28
Oscillatoria sp. PCC 7515, Dr . Ripp ka 1 29
Oscillatoria
sp . IRRI Farm 1 30
Scy tonema
sp. IRR I Farm , Upp er MN 1 1 1 31
Tolypothrix tenuis Dr. I . Watan abe, s t rain 1 1 1 1 32
Total 7 22 24 6
A: la bora tory m ass cul tures ; B: f lask cul tures less than 4 weeks old; C: f lask cul tures older than 4 weeks; D: soil -based inocula.
Reference numbe r s ar e those used in the Appe ndix for f l ask cul tures
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134 P.A. Roger et al. : Composition of blue-green algae
were covered with Parafilm' and incubated in da rkness at 30°C.
At time intervals, triplicate samples were taken and extract ed with
200 ml 2N KCI for 30 min with a wrist-action shaker. Exchangea ble
NH~-N was determ ined by st eam distillation with MgO.
esults and discussion
Laboratory flask cultures
Average values and variability.
Pooled numerica l
da ta from cul tures harvested before and af ter 4 weeks
of growth are g iven in the Appendix . Average va lues
and an analysis of the variabil i ty of the data are
pres ente d in Table 3. A graphic summ arizat ion of the
data is presente d in Fig. 1. Poole d data show ed that
dry ma tter averaged 3.72 and exhibited large varia-
t ions [coefficient of variat ion (CV) = 66 ] from
0.28 to 13.1 . Lower values wer e observe d in the
mucilaginous genera
Gloeotrichia,
and
Nostoc.
Non-
mucilaginous Calothrix and Fischerella had above-av-
erage values. Nitrogen content averaged 5 and
exhibited mo der ate variat ions (CV = 31 ) with
values ranging from 3.40 to 8.26. Compared with
o the r gene ra Anabaena had a higher N content
(6 .7 ) . Carbon content averaged 41 .6 and had the
lowest variabil ity (CV = 20 ). Carbon: ni trogen
rat io ave raged 8.09 and range d from 5 to 13. The two
ext reme values were observed in the same genus
Anabaena). Chlorophyll average d 0.7 and phyco-
cyanin 5.2 . Both exhibited a wide range of varia-
t ion as shown by coefficients of variat ion of 81 for
chlorophyll and 67 for phycocyanin.
To som e extent Fig. 1 and Table 3 al low a compari-
son be twee n intergen eric and intrageneric variabil ity
of the composit ion. The average value of the CV of
the five genera (average generic CV) is an est imate of
intrageneric variabil i ty whereas the CV of the mean
Table 3. Average generic values and variability of the conte nts in major compo nent s in N 2 -fixing BGA grown in flask cultures
Genera DM N C C:N Sugars Chl a Phyc Average a
CV
Ana bae na n 6 6 5 5 6 6 6
Mean 4.07 6.66 51.1 7.46 30.0 1.61 11.6
CV 73 28 20 42 28 46 35 39
Calo thri x n 9 9 3 3 9 9 9
Mean 6.26 5.38 35.6 6.61 56.4 0.62 3.62
CV 13 20 10 24 32 80 23 30
Fischerella n 4 4 3 3 4 4 4
Mean 6.48 3.85 35.6 8.09 43.3 0.48 2.40
CV 69 42 11 25 26 76 44 42
Gloeo trichia n 6 6 2 2 6 6 6
Mean 1.70 4.89 39.5 8.28 64.2 0.61 3.95
CV 94 36 10 24 41 67 49 46
Nostoc n 14 14 6 6 14 14 14
Mean 2.62 4.83 42.2 9.12 51.0 0.61 5.67
CV 61 26 10 34 26 71 38 38
Othe r genera n 7 7 3 3 7 7 7
Mean 3.81 4.10 nd nd 67.3 0.48 3.40
CV Not taken into account nd
Pooled data n 46 46 22 22 46 46 46
Mean 3.72 4.99 41.6 8.09 53.2 0.71 5.19
CV 66 31 20 31 37 81 67 48
Max. 13.1 8.26 67.3 13.2 114 2.45 15.5
Min. 0.28 1.78 29.7 4.82 19.9 0.08 1.14
Average gener ic CV 62 30 12 30 31 68 40 39 b.
CV of the meansc 48 20 16 12 26 59 67 35 a
Nitrogen (N), Carbon (C), sugars, chlorophyll a (Chl a), and phyco cyani n (Pcy) are expressed in percentage of dry weight. Dry matter
content (DM) is in percentage of fresh weight. Original data are presented in the Appendix.
n: number o f data: CV: coefficient of variation as percentage of the mean
bIntragenerie CV
Average intrageneric CV
e
dIntergenerlc CV
Average intergenerie CV
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P.A . Roge r et al . : Com posi t ion of blue-gree n algae 135
Dry matter Nitrogen Carbon
( f.w.) ( d.w .) ( d.w.)
C F G N P A C F G NA
46 4
::::::
ii 9 4
Red Sugars
I d.w.)
&
4
z
÷ li I
4
Chlorophyll a
( d.w.')
P A C F G N
sol~ l
i
50 ~ " i
30 -~:::: 3 .3
22
Phycocyanin
( d.w.)
p A C F
i
so ~ :i
I
40 :~x
~ 9 4
ZO
46 6
G N
P A C
2.0
o - ilili
1.5
If
°
O.5
r~
4
I
- .
15
1.0
A C F G N
ilill
iili lii
4t 4 6
Fig. 1. Gra phic summ arizat ion of the analysis of N2-fixing B GA grown in f lask cul tures .
Vertical bars
show ext r eme and average values for
the pooled dat a (P) and for each genus
(A: Ana baena ; C: Calothrix; F: Fischerella; G. Gloeotrichia, N N ostoc).
Number of s t r a ins
analyzed is indicated on the top or bottom of each bar
values of the five genera is an est imate of the interge-
neric variabil i ty. For dry matter, N, sugars, and
chlorophyll , average intrageneric variabili ty was lar-
ger than the intergener ic variabil i ty. For carb on and
phycoc yanin average intrageneric variabili ty was low-
er than the intergeneric variabil i ty (Table 3). This
was mostly due to a higher mean value and larger
variability of
Anabaena
strains which appear ed to be
clearly separated from the other genera (Fig. 1).
When al l variables are considered together (last co-
lumn, Table 3), i t appears that average intrageneric
variabili ty (39%) was sl ightly higher than intergene -
ric variability (35%).
Effect of the age of the culture.
The comparison of
cultures less than and older than 4 weeks (Fig. 2)
showe d a decrease in N and pigment contents and an
increase in sugars in older cultures. Results for dry
matter are variable but apparently there was a de-
crease in dry matter content in older mucilagenous
strains Anabaena, Gloeotrichia, Nostoc) and an in-
crease in nonm ucilageno us strains
Calothrix, Fische-
rella).
aumber fdata
Dry matter
Nitrogen
Sugarl
hlorophyll
O
Phycocyonln
Correlations between the constituents. When samples
less than 4 weeks old, for which C analysis was
perf orm ed, ar e consid ered (Table 4A ), a highly signi-
ficant posit ive correlat ion was obse rved betw een pro-
Fig. 2. Inf luence of age of cul ture on dry m at ter , N, sugars /chloro-
phyl l a , and phycocyanin content s . The numb er s under each s t r a in
are the number of da t a analyzed, Solid bars; cul tures less than 4
weeks old;
shaded bars:
cul tures more than 4 weeks old
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1 3 6 P . A . R o g e r e t a l. : C o m p o s i t i o n o f b l u e - g r e e n a l g a e
T a b l e 4 . C o e f f i c i e n t s o f c o r r e l a t i o n b e t w e e n t h e c o n t e n t s i n s o m e m a j o r c o m p o n e n t s o f N 2 - f i x in g
B G A
A . 2 2 c u l t u r e s l e ss t h a n 4 w e e k s o l d
n 2 2
P r o t e i n s + 0 . 3 1 n s r 1 % = 0 . 5 2
S u g a r s - 0 . 3 2 n s - 0 . 5 9 * * r 5 % = 0 . 4 1
C h l a - 0 . 0 2 n s + 0 . 7 2 * * - 0 . 7 1 *
P h y e o c y a n i n + 0 . 0 7 n s + 0 . 6 9 * * - 0 . 6 1 * + 0 . 6 1 *
C a r b o n - 0 . 4 2 * + 0 . 1 9 n s - 0 . 2 3 n s + 0 . 5 4 * *
D r y m a t t e r P r o t e i n s S u g a r s C h l a
B . 2 4 c u l t u r e s o l d e r t h a n 4 w e e k s
+ 0 . 3 4 n s
P h y c o c y a n i n
n = 2 4
P r o t e i n s - 0 . 2 9 n s r 1 % = 0 . 5 0
S u g a r s - 0 . 1 5 n s + 0 . 3 1 n s r 5 % - - 0 . 4 0
C h l a - 0 . 2 6 n s + 0 . 5 9 * * - 0 . 1 3 n s
P h y e o e y a n i n - 0 . 3 4 n s + 0 . 6 0 * * - 0 . 0 2 n s + 0 . 4 6 *
D r y m a t t e r P r o t e i n s S u g a r s C h l a
C . P o o l e d d a t a ( A + B )
n = 46
P r o t e i n s - 0 . 0 2 n s r 1 % = 0 . 3 7
S u g a r s - 0 . 1 3 n s - 0 . 1 9 n s r 1 % = 0 . 3 7
C h l a - 0 . 0 1 n s + 0 . 6 3 * * - 0 . 5 9 * *
P h y c o c y a n i n - 0 . 0 6 n s + 0 . 6 7 * * - 0 . 4 3 * * + 0 . 6 6 * *
D r y m a t t e r P r o t e i n s S u g a r s C h l a
* : s i g n i f i c a n t a t 5 % l e v e l; * * : s i g n i f i c a n t a t 1 % l e v e l
tein and pigm ent chlorophyll and phycocyanin) con-
tents. A highly significant negative correlat ion was
observed between sugar and pro te in conten ts , and
between sugar and p igment conten ts . A negat ive
corre la t ion was observed between C conten t and dry
mat te r bu t no t be tween sugar conten t and dry mat ter .
The re was no significant correlat ion betw een carbon
and sugar contents.
Negat ive corre la t ions observed between sugars
and proteins or pigments in young cultures appear to
be age related because th ey beca me nonsignificant in
older cultures Table 4B).
Poo led data Table 4C) showed a posi t ive correla-
t ion be tween p ro te in and p igm ent conten ts and ch lo-
rophyll a and phycoc yanin contents, and a negative
correlat ion betw een sugars and pigme nt contents. No
corre la t ion was found between sugars and pro te in
contents.
Dendrogram analysis
Dend rogram analysis o f pool-
ed data Fig. 3) shows a division into two majo r
groups 1-33 and 34-46). The first group contains al l
strains older than 4 weeks and some strains younger
than 4 weeks. The second group comprises strains
younger than 4 weeks. Furthe r grouping of the s t ra ins
shows some tendencies but no rigorous grouping of
the species. The following groups can be distin-
guished: 1-10, dominated by mucilaginous strains
older than 4 weeks; 11-22, domin ated by nonmucila-
ginous strains but the re is no clear effect of the age of
the culture; 23-33, d om inate d by culture older than 4
I A
N
5 N
6 N
7 G
8 N
S N
10 N
II C
12 C
13 N
~ T
15 N
6 N
17 F
8 C
2O F
21 0
2 2 C
23 Ap
24 G
2 5 N
26 S
27 C
28 C
29 C
~ T
31 N
32 G
3 3 F
3 4 A
S 5 A
~s AN
37
3 8 /
39 A
4 a c
41 N
42 C
43 g
4 4 F
45 G
4 6 O
I
0
older than 4 weeks
[ ] younger than 4 weeks
Y~/ mucilaginous strain
~:i: :
nonmuci loginous stra in
[ ]
A n a b a e n a
t
I I I I I I I I
I 3 4 5 6 7 8
F i g . 3. D e n d r o g r a m a n a l y s i s o f t h e c o m p o s i t i o n o f t h e f l a s k
c u l t u r e s
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P . A. Roge r e t a l . : Com pos i t i on o f b lue - g r een a lgae
1 A
2 A
3 N
4 A
5 A
6 A
? Au
8 F
9 C
10 N
11 N
1Z T
13
N
14 C
15
N
16 0
17 C
18
F
19
F
2O G
Z1 G
22 N
I ~ mul:itatlnolli siroln I
~ii ii ni~,nucllaglt-~ stroln
i ~ ] A n o b a e n o
- - I
3
1786
1Z86
1 I I I 1 I I
0 1 2 3 il 5 6 7
Fig . 4 . Dend r ogr am ana lys i s o f t he compos i t i on o f f l a sk cu l tu r es
l e ss t han 4 weeks o ld
®
PC 2
ll~fli.ltrll::e of l ie
~alum of 1*~
s lfoin
] nlui:iloltlnot,ls lll"oll~
no~ nliciloginous strain
Al~ooena spp
~D
[ ]
©
~ 3 4 PCl
2
©
_ © i lr~©
r ~ N g N
[ ] - -3
]
]
d~
Fig . 5 . P r inc ipa l compo nen t s ana lys i s o f t he compos i t i on o f f l a sk
cu l tu r es : d i s t r i bu t ion i n sco r es and n a tu r e o f t he s t r a in . A: Anabae
na; Ap: Aphanathece; Au: Aulosira; C: Calothrix; F: Fischerella;
G: Gloeotrichia; N: Nostoc; O: Oscillatoria; S: Scytonema; T:
Tolypothrix
137
weeks, both mucilaginous and nonmucilaginous
strains are included; 34-39,
Anabaena
more than 4
weeks old; 40-46, cultures less than 4 weeks old.
W hen cultures less than 4 weeks old, for which 5
variab les were taken in to account for dendrogram
establishment (Fig. 4), are considere d, only a group-
ing of Anabaena strains is obvious (1-6); other gene-
ra are spli t between the two other subgroups (7-13
and 14-22).
Multivariate analysis. Principal component analysis
of the pooled data (Table 5) shows that the first two
components represented about 74 of the to tal vari -
ance. Th e first com pone nt had ma jor values of load-
ing on N and pigment contents (posi t ive) and sugar
conten t (negat ive) . The second component had ma-
jor loading on dry m atter con tent (posi t ive) and sugar
content (negative). When the scores of the strains
and their nat ure (Fig. 5) are considered, three over-
lapping clouds can be distinguished: Anabaena
strains, mucilaginous strains, and nonmucilaginous
strains. W hen the scores of the strains according to
their age (Fig. 6) are con sidered, a grouping into two
overlapping clouds and a larger variabil i ty of the
composit ion of young strains compared with old
strains is obvious.
Laboratory mass cultures
The six strains used
Anabaena variabilis, Aulosira
fertilissima, Fischerella sp.,Scytonema sp., Nostoc
sp. , and Tolypothrix tenuis) consti tute a representa-
t ive sample of the s t ra ins frequent ly recomm ended
for inoculat ion in rice fields (Venkataraman 1981).
W hen grow n under art ifical condit ions and harves ted
at the end of the logarithmic phase of grow th , these
strains had an N con tent , on an ash-free basis, averag-
ing 8 and exhibiting relatively low variation s
amon g strains (CV -- 9 ) (Table 6). The carbon
conten t averag ed 45 (ash-free basis) and had a
rem arkab ly low range of variat ion (CV -- 4.6 ). The
carbon: ni trogen rat io ranged from 4.8 to 6.3 and
T a b l e 5 . P r i n c i pa l c o m p o n e n t s a n a l y s i s o f t h e c o m p o s i t i o n o f t h e f l a s k c u l t u r e s a n d l o a d i n g f a c t o r s
(n = 46)
Dr y Ni t r ogen Sugar s Ch l a Phyco- o f Cum ula t e
m a t t e r c y a n i n v a r i an c e o f
va r i ance
1 0 . 005 0 . 488 - 0 . 405 0 . 557 0 . 536 52 . 4 52 . 4
2 0 . 877 - 0 . 225 - 0 . 401 - 0 . 028 - 0 . 135 21 . 5 73 . 9
L oad ing 3 0 . 472 0 . 541 - 0 . 675 - 0 . 111 0 . 129 15. 3 89 . 3
f ac to r s 4 0 . 044 - 0 . 276 - 0 . 051 - 0 . 552 0 . 784 6 . 5 95 . 8
5 0 . 081 - 0 . 585 0 . 465 0 . 610 0 . 250 4 . 2 100
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138
P.A. Roger et al.: Composition of blue-green algae
• • •
IJ • •
• l u •
° l •
l o° •
• • • o m
Fig 6. Principal compon ents analysis of the compo sition of f lask
cultures: dis tr ibutio n in scores and age of the strain influence of the
age of the culture: • - olde r than 4 weeks: • - y ounger than 4
weeks
averaged 5.6. Ash content averaged 7.5 and rang-
ed from about 6 to 12 . Avera ge cation contents
(P, K, M g, Ca) were o f the same order of magnitude,
ranging from 0.55 to 0.65 . Variability was higher
than that of C and N. Highest variability was observ-
ed for Mg content. Concentrations of oligoelements
(Cu, Mn, Zn, Fe, A1, Na) ranged from a few ppm
(Cu) to 2000 ppm (Fe). All exhibited a very high
degree of variation (C V = 69 ) whereas other oli-
goelements exhib i ted modera te var ia t ion (CV =
2 3 - 2 9 ) .
Blooms produced on soil in trays
Algal b loom s produced on so i l had average conten ts
of 26.7 ash, 6.3 N and 42.8 C on an ash-free
basis (Table 7) . The lowest variabil i ty was observed
Table 6. C ompositi on of mass cultures o f BGA harvested at the end o f the logarithmic phase of growth. Data are expressed on a dry
weight basis
Ash N N ash C Ca sh C:N P K Mg
free free
( ) ( ) ( ) ( ) ( ) ( ) ( ) ( )
Ca Cu Mn Zn Fe A1 Na
( ) (ppm) (ppm) (ppm) (ppm) (ppm) (ppm)
Anabaena
6.48 7.88 8.42 40.8 43.6 5.18 0.62 0.59 0.54 0.60 18 605 62 1586 156 0.82
variabilis
Aulosira
5.65 7.62 8.08 41.9 44.4 5.50 0.61 0.48 0.48 0.49 34 545 83 1414 188 0.58
fertilissima
Tolypothrix
6.14 6.92 7.37 41.6 44.3 6.01 0.53 0.47 0.39 0.60 23 505 44 1416 611 0.80
tenuis
Scytonema
sp. 8.85 6.29 6.90 39.7 43.5 6.31 0.40 0.65 0.44 0.68 24 561 59 2085 963 1.48
Fischerellasp 11.80 7.04 7.98 41.1 46.6 5.84 0.75 0.57 1.09 1.02 22 925 92 2345 1630 0.93
Nostoc
sp. 7.50 8.55 9.24 41.1 44.4 4.81 0.66 0.63 0.48 0.67 17 677 42 1845 904 1.04
Anabaena 5.58 7.68 8.13 44.6 49.3 5.81 0.56 0.46 0.45 0.49 31 431 51 1250 664 0.47
7120
Mean 7.42 7.42 8.02 41.5 45.1 5.64 0.59 0.55 0.55 0.65 24 607 66 1705 731 87
CV 30.2 9.93 9.31 3.6 4.6 9.1 18.6 14.4 43.6 27.7 26.1 26.3 29.7 23.5 69.2 28.6
CV: coefficient of variation as a percentage of the mean
Table 7. Composition of blooms produced in soil trays in a greenhouse
Strain
Gloeotrichia Aulosira Anaba ena Cylindrosp ermum
Average CV
( )
Remarks Average of two One
composite composite One sample One sample
samples (2 X 4) samples (4)
Dry matte r ( fresh weight) 0.94 6.41 2.45 6.28 4.02 68
Ash ( dry weigh t) 27.3 27.5 15.0 37.2 26.75 34
N ( dry weight ) 3.01 5.83 6.44 4.13 4.85 32
N ( dry weight, ash free) 4.00 7.34 7.43 6.29 6.26 25
C ( dry weigh t) 30.14 31.1 39.4 28.8 32.36 15
C ( dry weight, ash free) 40.0 41.9 45.4 43.8 42.8 5
C:N 10.0 5.3 6.1 6.9 7.07 29
P ( dry weight) 0.14 0.35 0.39 0.38 0.31 37
K ( dry weight) 0.32 0.39 0.47 0.19 0.34 35
Mg ( dry weight ) 0.43 1.50 0.43 0.31 0.64 79
Ca ( dry weight ) 1.53 2.19 2.62 1.85 2.05 23
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P.A. Roger et al.: Composition of blue-green algae
for C (CV = 5 ). Coefficients of variat ion were 34
for ash and 25 for N.
Monospecific soil based inocula
Soil-based inocula were characterized by very high
ash contents (78.4 -81.3 ), obviously due to the
soil harvested with the algal material (Table 8).
Nitrogen in ash-free algal material , calculated from
the N and ash contents in the harves ted material and
in the soil ut i l ized for mult ipl icat ion, ranged from
5.9 to 8 . Nitrogen conten t of the standing bio-
masses was eq uivale nt to values ranging from 11 to 19
kg N/ha.
Field samples
General results.
Analysis of 11 samples of field-grown
BGA is presented in Table 9. Ash content was high
and averaged 52 . Nitrogen content on an ash-free
139
basis averaged 4.8 . Carbo n conten t on an ash-free
basis averaged 40 and exhibited low variability
(CV -- 7 ). The carbon: nitrogen ratio averaged 8.5
and ranged from 6.6 to 11.6. The p hosphorus content
averaged 0.1 and K 0.3 . Because of the high ash
content of the samples, Mg and Ca were high and
exhibited large variability.
Detailed analysis of a mucilaginous bloom. Table 10
presents the analysis of a bloom of mucilaginous
Nz-fixing strains which has been observed yearly
since 1980 in non-N plots of IRRI's farm during the
dry season. It consisted of
phanothece
s the domi-
nant genus and
Gloeotrichia
s an associated one.
The material was characterized by low dry matter
conte nt (1.31 ), high ash conten t (59 ), and low N
conte nt even on an ash-free basis (3.8 ).
Although the colonies were careful ly washed and
drained, collected material had a high ash content.
This indicates that mucilage of the algae absorbs soil
Table 8. Compositio n and productivit y of monospecifi c soil-based inocula of N 2 -fixing BGA
Soil-algal mat BGA (ash flee)
Strain Dry weigh t N ( ) C ( ) Ash ( ) Dry weigh t Algal N N
(g/m 2 ) (kg/ha) (kg/ha) (kg/ha)
Soil before - 0.150 1.33 84.4 - - -
inoculation
Anabaena variabilis 313 0.509 3.78 78.5 176.0 15.94 6.32
Aulosira fertilissima 470 0.545 3.92 79.0 278.6 13.24 7.03
Fischerella sp. 273 0.758 4.73 78.4 212.5 13.29 5.88
Nostoc sp. 377 0.563 4.25 79.3 252.1 11.50 6.53
Scytonema sp. 430 0.444 3.24 81.3 188.3 18.98 6.81
Tolypothrix tenuis 356 0.514 3.92 79.8 226.2 16.91 7.96
Table 9. Analysis of field samples of N 2-fixing BGA (all data in perce ntage dry weight)
Sample Location Ash N N ash C C ash C:N P K Mg Ca
free free
Aphanothece Nostoc Nueva Ecija 46.1 2.83 4.89 26.1 45.1 9.2 0.122 0.343 1.22 8.30
bloom
Nostoc commune Luzon 30.7 3.20 4.49 29.7 41.7 9.3 0.050 0.172 7.49 1.18
Mixed algal flakes India 64.4 2.34 5.97 15.4 39.2 6.6 0.121 0.271 0.45 2.71
Aphanothece Nostoc IRRI 71.3 1.62 4.76 12.8 37.8 7.9 0.113 0.320 2.25 6.13
bloom
Aphanothece bloom IRRI 43.8 2.49 4.11 23.1 38.1 9.3 0.181 0.569 2.07 3.95
Nostoc bloom IRRI 55.9 2.76 5.63 18.9 38.7 6.9 0.159 0.475 1.56 2.10
Nostoc bloom IRRI 45.6 2.44 4.15 21.4 36.6 8.8 0.081 0.350 3.11 7.77
Aphanothece Gloeotrichia IRRI 58.8 1.75 3.82 20.4 44.5 11.6 0.074 0.388 3.29 7.20
bloom
Nostoc bloom IRRI 55.2 2.64 5.32 18.9 37.6 7.1 0.142 0.285 1.92 1.04
Aphanothece bloom IRRI nd 2.93 nd nd nd nd nd nd nd nd
Nostoc commune colon ies Mangatarem 49.6 2.72 4.99 21.4 39.3 7.9 0.109 0.122 5.23 1.16
Mean 52.1 2.52 4.81 20.8 39.9 8.5 0.115 0.329 2.86 4.15
CV 22 19 14 23 7 18 34 40 73 70
Maximum 71.3 3.20 5.97 29.7 45.1 11.6 0.181 0.569 7.49 8.30
Minimum 30.7 1.62 3.82 12.8 36.6 6.6 0.050 0.122 0.45 1.04
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140
Table 10. Analys i s of a b loom of muci l aginous s t ra ins
With ash Ash f ree
Dry mat t er 1 .31 ns
Ash ( ) 58.8 ns
C ( ) 20.4 44.5
N ( ) 1.75 3.82
C:N 9.93
P ( ) 0.07 0.16
K ( ) 0.40 0.85
Mg ( ) 3.29 7.18
Ca ( ) 7.20 15.7
Na ( ) 2.03 4.42
Fe ( ) 1.35 2.94
AI ( ) 2.20 4.78
Cu (ppm) 30.3 66.1
Mn (ppm) 705 1535
Z n ( ppm ) 52 114
B (ppm) 641 1397
Chlo rophy l l a ( ) 0.15 0.33
Phyco cyanin ( ) 1 .36 2 .94
aDry mat t er i s expres sed on a f r esh weight bas is ; o ther da t a
are expres sed on a d ry ma t t er bas i s
Nmineralzed
8
~ Bact~ia3.5)
~ 0 ~ AzolIo1
_0)
Straw591
0
I
[
I I
0 5 I0 15 2
Doys
Fig. 7 . Ni t rogen minera l i za t ion r a tes of d i f fer ent BGA compared
with bacter ia,
Azolla
and s t raw.
Figures
in
parenthesis
are C/N
values
part icles. Th ere fore , values presen ted in Table 10 for
major ca t ions and o l igoelements do not show the
comp osit ion of the algae but that of a m ixture of algal
material and soil part icles. Soil where the bloom
deve loped had 84.4 ash, 1.23 C, and 0.13 N in
the upper horizon. Assuming a 40 C conten t in
ash-free algal mater ial and 7 ash in algal material
free of soil , and using values from Table 1 for ash and
C con tent , the co ntent o f soil part icles in the harvest-
ed mater ial is about 60 of the dry weight .
P.A. Roger et al . : Composi t ion of blue-green algae
A similar calculat ion from ash and N con tents from
Tab le 1, assum ing 7 ash in algal ma terial free of soil
particle , led to a similar value of abou t 61 of soil
part icles in the h arvested m aterial .
The h igh boron conten t o f the harvested mater ia l is
in agreem ent with the boron toxicity repo rted at the
IRRI fa rm.
Mineralization study
Results of the mineral izat ion study (Fig. 7) show a
clear correlat ion between the C:N rat io of the mate-
rial and the percentage of N mineral ized at a given
time. Depending on the C:N rat io of the strain,
be tween 30 and 65 of BG A ni t rogen was minera l-
ized in 3 weeks.
eneral discussion
Components
Dry matter.
Dry mat ter conten t in labora tory-grown
strains averag ed 3.7 . T he highest values were re-
corded for a Fischerella strain (13 ) and an Anabae-
na
strain (8.5 ). T he lowest values were recorded
for m ucilaginous strains of
Nostoc
(0.28 ) and
Ap-
hanothece
(0.30 ). In blooms prod uced on soil , the
average value was close to that of laboratory strains
(4.02 ). The highest value was recor ded for
Aulosi-
ra
(6.4 ) and the lowest for a mucilaginous
Gloeotri-
chia
(0.94 ).
A few values recor ded from field samples showed
that species forming mucilaginous colonies of a def-
ini te shape
Aphanothece, Nostoc, Gloeotrichia)
usually have a low dry matter content of about
1 -2 . R oger and Kulasooriya (1980) repo rted
values of 2.2 for
Nostoc
and 0.74 for
Gloeotrichia
spp. Mucilaginous BG A can develop very impressive
blooms, but the corresponding dry matter is low.
Ro ger et al . (1985) re cord ed blooms of
Aphanothece
and Nostoc ranging from 7 to 33 t /ha fresh weight ,
corresponding to 74-132 kg/ha dry weight and
1.5-2.6 kg N/ha only.
Another characterist ic of dry matter content is i ts
large variabil i ty, as shown by coefficients of variat ion
of 50 -80 (Table 11). The variabil i ty is due part ly
to the natu re o f the strain. Mucilaginous gener a have
less dry matter than nonmucilaginous ones. How-
ever, larger variabil i ty was also observed between
species of the s ame genus. F or exam ple, values rang-
ing from 1.0 to 8.5 have been recor ded for
Anabaena
and values ranging from 0.28 to 6.3
have been recorde d for Nostoc (Append ix). In labora-
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P.A. Roger et al.: Composition of blue-green algae 141
Table 11. Su mmarizat ion of the data on the c ompositio n of N 2-fixing BGA. Data are expressed as percentage of dry weight exc ept dry
matter ( fresh weight) and C:N
Dry Ash N N C C C:N P K Mg Ca
matter (ash (ash
free) free)
Data from Mean na 6- 7 7.0 7.5 a 43.0 46.0 a 5.4 0. 96 na na na
the lit era tur e n - 2 24 24 5 5 5 9 - - -
n variable Max. - - 11.0 11.8 48.0 51.6 7.4 2.10 - - -
Min. - - 2.8 3.0 38.0 40. 9 4.3 0.1 8 - - -
CV - - 34 34 12 12 22 74 - - -
Laborat ory mass Mean nd 7.4 7.42 8.02 41.5 45.1 5.6 0.59 0.55 0.55 0.56
cultur es at expo- Max. - 11.8 8.55 9.24 44.6 49.3 6.3 0.75 0.63 1.09 1.02
nent ial phase of Min. - 5.6 6 .22 6.90 39.7 43.5 4.8 0 .40 0.46 0.39 0.49
gro wth , n -- 7 CV - 30 10 9 4 5 9 19 14 44 28
Labo rat ory flask Mean 3.85 nd 5.48 5.89 a 41.6 44.7 a 8.1 nd nd nd nd
cultures less than Max. 8.50 - 8.26 8.88 67.3 72.0 13.0 . . . .
4 weeks old Min. 0.28 - 3.40 3.65 31.9 34.3 4.8 . . . .
n = 22 CV 54 - 28 28 20 20 31 . . . .
Laborato ry flask Mean 3.56 nd 4.32 4.64 nd nd nd nd nd nd nd
cultures older Max. 13.64 - 8.02 8.60 . . . . . . .
than 4 weeks Min. 0.30 - 1.79 1.92 . . . . . . .
n = 24 CV 83 - 34 34 . . . . . . .
Artifi cial blo oms Mean 4.02 26.7 4.85 6.26 32.4 42.8 7.1 0.31 0.34 0.64 2.05
prod uced on soil Max. 6.41 37. 2 6.44 7.43 39.4 45.4 10.0 0.39 0.47 1.50 2.62
four comp osit e Min. 0.94 15.0 3.01 4.00 28.8 40.0 5.3 0.14 0.19 1.31 1.53
samples CV 68 34 32 25 15 5 29 37 35 79 23
Field samples Mean nd 52.1 2.52 4.81 20.8 39.9 8.5 0.12 0.32 2.86 4.15
n = 11 Max. - 71.3 3.20 5. 97 29.7 45.1 11.6 0.18 0.57 7.49 8.30
Min. - 30.7 1.62 3.82 12.8 36.6 6.6 0.05 0.12 0.45 1.04
CV - 22 19 16 23 7 18 34 40 73 70
Pooled values
Mean 3.28 27.6 5.34 6.03 36.3 43.7 7.71
Max. 13.64 71.3 11.00 11.8 67.9 72.0 13.0 2.10 0.63 7.49 8.30
Min. 0.28 5.6 1.62 1.9 12.8 36.6 4.3 0.05 0.12 0.39 0.49
aExtr apola ted on the basis of 7 ash conten t
t o r y c u l t u r e s , a p p a r e n t l y t h e r e w a s a d e c r e a s e i n d r y
ma t t e r c o n t e n t i n o l d e r mu c i l a g i n o u s st r ai n s a n d a n
i n c r e a s e i n n o n mu c i l a g i n o u s o n e s . T h e r a n g e o f v a r ia -
t i o n o b s e r v e d f r o m p o o l e d d a t a (0 . 1 8 - 1 3 . 6 4 )
s h o w s t h a t t h e f r e s h w e i g h t o f a s t a n d i n g b l o o m o f
BG A i n a r ic e f i e ld d o e s n o t g i v e a n y i n f o r ma t i o n
abou t i t s ag ronom ica l s ign if i cance .
A s h
L a b o r a t o r y ma s s c u l t u r e s h a d a n a v e r a g e a s h
c o n t e n t o f 7 . 4 , r a n g i n g f r o m 5 . 6 t o 1 1 . 8 , w h e -
reas f i e ld samples , cons i s t ing ma in ly o f muc i l ag inous
s t ra i n s, h a d a n a v e r a g e a s h c o n t e n t o f 5 2 , r a n g in g
f r o m 3 1 t o 7 1 . B l o o ms p r o d u c e d i n t r a y s , w h e r e
t h e r e w a s m i n i m u m d i s tu r b a n c e a n d w h e r e d e m i n e r -
a l iz e d w a t e r w a s u se d , h a d l o w e r a s h c o n t e n t s ( 1 5 -
37 ) tha n those g rown in the f i e ld , ind ica t ing tha t , as
w i t h mo s t a q u a t i c p l a n t s , a s h c o n t e n t o f BG A i s
re l a t e d to the quan t i ty o f so il par t i c l es in suspens ion
i n t h e f l o o d w a t e r ( Ro g e r a n d W a t a n a b e 1 9 8 4 ) . A s h
c o n t e n t ma y b e e s p e c i a l l y h i g h i n s h e a t h - f o r mi n g
s t ra ins tha t m ay adsorb c l ay and s il t i n the i r muci l age .
T h e w i d e r a n g e o f a s h c o n t e n t o b s e r v e d i n f i e l d
s a mp l e s ( 3 1 - 7 1 ) s h o w s t h a t t h e d r y w e i g h t o f a
s t and ing b loom of BGA in a r i ce f i e ld g ives l i t t l e
in fo rmat ion abou t i t s ag ronomic s ign i f i cance .
Protein and nitrogen
W h e n t h e r e s u lt s w e r e e x p r e s s-
e d i n p e r c e n t a g e d r y w e i g h t , a l a r g e d i f f e r e n c e i n
a v e r a g e N c o n t e n t b e t w e e n l a b o r a t o r y c u l t u r e s
( 5 . 2 ) a n d f i el d s a mp l e s ( 2 . 5 ) w a s o b s e r v e d . Be -
c a u s e o f t h e d i f f e r e n c e i n a sh c o n t e n t o f t h e ma t e -
r i a l s , c o mp a r i s o n s w e r e ma d e o n a s h - f r e e ma t e r i a l .
N i t r o g e n c o n t e n t r a n g e d f r o m 1 . 9 t o 1 1 .8 a n d
a v e r a g e d 6 o f a s h - f r e e d r y w e i g h t .
I n t h e l i te r a t u r e , d i f f e r e n t v a lu e s h a v e b e e n r e p o r t -
e d f o r t h e a v e r a g e p r o t e i n o r N c o n t e n t in BG A : 5 0
pro te in o r 7 .9 N (Fogg e t a l. 1973) , 37 -66
p r o t e i n o r 5 . 8 - 1 0 . 4 N ( Mi s h u s ti n a n d Sh i l' n i-
kova 1971), and 20 -45 p ro te in o r 3 .2 -7 .1 N
(Holm-Hansen 1968) . Wolk (1973) s t a t ed tha t so
long as N i s ava i l ab le in ab und an t and eas i ly as simi l -
a b l e f o r m a n d t h e a l g a e a r e n o t p r o d u c i n g c o p i o u s
a mo u n t s o f mu c i l a g e , N a c c o u n ts f o r a b o u t 1 0 o f
c e ll d r y w e i g h t. T w e n t y - f o u r N m e a s u r e me n t s i n
N 2 - f i x i n g BG A r e c o r d e d f r o m t h e l i t e r a t u r e r a n g e
f r o m 3 t o 1 1 a n d a v e r a g e 7 . 0 2 ( T a b l e 1 ). T h e
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142
P.A. Roger et al.: Compos ition of blue green algae
variabil i ty of the est imates for the average protein
conten t o f BG A is most p robably due to the fac t tha t
only few data w ere cons idered in the calculations. In
addit ion, most of these data refer to laboratory
strains, which may have led to biased est imation.
Labo ra tory s t ra ins are frequent ly grown in op t imized
condit ions and, usually, under l ight intensi t ies muc h
lower than those in natural environments. Within
certain l imits, an inverse correlat ion between l ight
in tensi ty and photopigm ent conten t i s genera l among
photosynthet ic organisms; BG A are no except ion to
this rule (Cohe n Bazire and Bryant 1982). Ther efor e,
one can expect a h igher p igment conten t and lower
ash content in laboratory-grown material , which
leads to a higher relat ive protein content .
The protein and N contents in BGA exhibit varia-
t ions related with the na ture of strains, the physiolog-
ical state of the culture (mainly its age), and environ-
ment . High pro te in conten ts were reported for
An
abaena cylindrica
(43 , Collyer and Fogg 1955) and
Aphanothece halophitica
(76 , Tindal et al. 1977)
and som e non Nz-fixing species such as
Spirulina
spp.
(62 -73 ) . On the o ther hand , a va lue lower than
30 has been reported for
Calothrix
sp. (Williams
and Bur ris 1952). O ur results did not al low us to find
out if some species or genera exhibited consistently
h igher N conten ts than o thers . The h ighest average
value observed wi th
Anabaena
strains (Table 4) was
probably because most o f the cu l tures analyzed were
less than 4 weeks old.
From the ranges of variat ions among strains (Fig.
1) i t appears that intrageneric variabil i ty of the N
conte nt is large, as shown by the coeff icient of varia-
t ion ranging from 20 to 42 and averaging 30
(Table 4). D ata from the l i teratu re give l i tt le informa-
t ion about intragener ic variabil i ty but show that intra-
specific variabil ity may be high. For exam ple, protein
conten ts ranging from 35 to 63 were reported for
Anabaena cylindrica (Table 1).
Large variat ions have been observed during the
growth cycle of some strains. N itrogen co ntent of A.
cylindrica
increased d uring the first stages of growth,
from about 5 at 2 days to about 10 at 11 days
(Cobb and M yers 1964). The highest protein content
of an axenic culture o f Apanothece stagnina (4 0 -
46 ) was obtained at the end of the lag phase and the
beginning of the exponent ia l phase (3rd-8 th day)
(De Cano and De Halperin 1978). The general ten-
dency for the pro te in conten t o f BG A to decrease as
the culture ages is clear, and was repo rted fo r natural
populat ions of
Aphanizomenon flos aquae.
Such po-
pulat ions containe d 30 -42 protein, with the hig-
hest p ro te in conten ts observed a t b looming (Shnyu-
kova et al. 1978). Our results also showed a decrease
in N con tent in aging material . This was observed in
laboratory cultures (Fig. 2) and in artificial and nat-
ural blooms. In laboratory cultures average N con-
tent was 8.02 in cultures at the exponential phase of
grow th, 5.89 in flask cultures less than 4 wee ks old,
and 4.64 in cultures mo re than 4 weeks old. Aver-
age N content in ash-free dry ma tter was 6.76 in
soil-based inocula harvested 2 weeks after inocula-
t ion, 6.3 in blooms prod uced on soil which were
harve sted 4 week s after submersion, and 4.8 in
natural samples which comprised a high proport ion
of mucilaginous strains harv ested a fter several weeks
of growth.
Pigments.
Phyco cyanin and chlorophyll a were mea-
sured in flask cultures only. Q uanti tat ively, bi l ipro-
te ins are the m ost importan t p igments in BGA . They
usually represent 1 -10 of the dry weight of the
cell (Cha pm an 1973) al though under certain circum-
stances i t ma y be more : 18.4 in
Tolypothrix tenuis
grown under fluorescent l ight (Hattori and Fuji ta
1959).
Usually phycocyanin is the major bi l iprotein in
BG A. I t may account for as much as 40 of the to tal
protein in fi laments of
Anabaena cylindrica
(Fay
1969). In th e studied samples, phyc ocyanin conten t
rang ed from 0 to 15.5 dry weight . Samples less
than 4 week s old had a higher conte nt (6.59 ) than
older ones (3.90 ). Similarly a decrease was observ-
ed for chlorophyll , from 1.11 to 0.75 . Aver age
and extreme values were within the range of values
reported in the l i terature (Table 1).
Phosphorus. The P level in BGA may fluctuate
widely depending on whether or no t the a lgae are
growing under P-l imited condit ions. Blue-green al-
gae assimilate mo re P than the y require and store the
excess as polyphosphate which can be used in P-defi-
cient condit ions (Batterton and Van Baalen 1968).
From Table 1 it appears that P content in N2-fixing
BG A rep orted in the l i te ra ture var ies f rom 0 .1 to
2 .2 . Larger va lues (0 .8 -4 .2 ) have been report -
ed for BG A in general by Mishustin and Shil 'nikova
(1971),who did not indicate the origin of their data.
Data summarized from the l i te ra ture co l lec ted by
Hea ley (1982) suggest that P con tent in BGA grow-
ing in a P-sufficient me dium averages 1.38 (highest
value 1.56 , lowest 0.75 , 9 data), whereas P con-
tent in BG A growing in P-deficient med ia could be as
low as 0.03 . Our strains grown on GO me dium as
mass cultures were comparatively deficient in P as
indicated by contents ranging from 0.4 to 0.75
and averaging 0.59 . A n accurate evaluation of P
content in BGA grown on soil or field samples is
impossible because of the high ash (soil) content of
the algal material . B ecause P conten t is usually lower
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P.A . Rog e r e t a l .: Com p os i t ion of b lue -gre e n a lga e
143
to
T
o
V
o ~ ~
•
05 1.0 1.5
P
Fig . 8 , Cor re la t ion b e twe e n N a nd P c onte nt s a sh- f r e e ba s i s) in
N: - f ix ing BGA : , 7 Da ta f rom the l it e r a ture : v l a bora tory m a ss
cultures : o ar t i f ic ia l blooms on soi l ; • f ie ld samples
in soi l than in the BG A mater ia l, noncor rec ted va lues
are underest im ated , whereas va lues cor rec ted fo r ash
content are overestimated because all the P contain-
ed in the mixture of BG A and soil is attr ibuted to the
BGA. Even when va lues cor rec ted fo r ash are con-
sidered, i t appears that according to Healey 's data,
ar t if icial blooms produced on soil (where P was
applied basally at a rate of 20 kg/ha) wer e deficient in
P, as indicated by an average content of 0.42% P on
an ash-free basis (Healey 1982). A similar observa-
tion was made with f ield samples which exhibited an
average P cont ent of 0.25% on an ash-free basis.
Figure 8 shows a highly significant positive correla-
t ion be tween N and P con ten ts expressed on an
ash-free basis. The general shape of the curve shows
that at P concentrations higher than 1% th ere was no
mo re increase in N content, indicating a luxury
consumpt ion in P. The op t imal va lue o f 1% was
at ta ined in labora tory cu l tu res on ly . BG A grown on
soil and natural samples had concentrations lower
than 0.5%, confirming that P availabil i ty is a major
limiting factor for BGA growth in natural environ-
ments (Roger and Kulasooriya 1980).
Carbon.
In cultures at the exponential phase of
growth, average C content was 45% of ash-free dry
mat ter and exhibited very low variabil i ty (CV = 5% ).
In f lask cultures, the average value was similar (45%)
but the variabil i ty was greater (CV = 20%). In
artif icial and natural blooms, C content was sl ightly
lower than in labora tory cu l tu res (40%-43%) and
var iab i l i ty was low (CV = 5%-7%). Average C
content in laboratory cultures (45%, 29 data) was
very close to that calcu lated from data collected from
the l i terature (46%, 9 data) . The lower C content in
artif icial and natural bloom s may be due to P deficien-
cy in these materials. He ale y and Hendze l (1975)
repor ted C con ten ts o f 45% -48% in P-suff ic ien t
(1.56% P in the cell) and slightly P-deficient (0.70%
P in the cell) Anabaena variabilis. In a strongly
deficient medium, cellular P was 0.13% and C con-
ten t 38%. However , more da ta a re needed to d raw
definite conclusions. A majo r characterist ic of C in
N2-fixing B G A seem s to be a low variabil ity compar-
ed with other elements. Therefore, an increase in
C:N ra t io observed when BGA mater ia l i s ag ing is
mostly due to a decrease in N content.
Carbohydrates.
Genera l ly , the percen tage of carbo-
hydrates found in N2-fixing BGA is very variable.
The mean from 11 reports (Table 1) is 38%. Values
range f rom 13% to 85%. The carbohydra te con ten t
of BG A var ies wi th the na ture and age of the o rga-
nism as well as with some environmental factors
(Mehta and Vaidya 1978). Polysaccharides are the
most common carbohydra tes in BGA. They are a
principal consti tuent of the cell wall and envelopes
that form during the d ifferentiation of heterocy sts
and spores. The cells of certain B G A characterist ical-
ly form mucilaginous capsules which are polysac-
charide in nature. Produ ction of extracellular polysac-
charides dep ends on the age of culture, the grow th
tem pera ture, and the form of N available (Sangar
and Dugan 1972). Extracellular mucilage can ac-
count for as much as 44% o f the dry weight of cultures
(Moore and Tischer 1964; Wolk 1973).
Po lysacchar ide con ten t , expressed as sugars , was
studied only in laboratory cultures grown in f lasks.
Values recorded for individual samples ranged from
20% to 114% and averaged 53%. The highest value,
larger than 100%, clearly demonstrates that there are
problems in sugar determination. Similar observa-
tions were made by Collyer and Fogg (1955), who
poin ted ou t tha t som e h igh va lues encountered in the
li terature may be due to methodological ar t ifacts
inherent to the phenol-sulfuric acid method.
Avera ge gener ic va lues (Table 4 ) ranged f rom 30%
Anabaena) to 64% Gloeotrichia). These was no
clear-cut difference between mucilaginous colony-
forming genera Nostoc, Gloeotrichia) and the o ther
genera . The lower va lue recorded for
Anabaena
s t ra ins i s p robab ly because most o f the samples were
less than 4 week s old. Our results showed an increase
in sugar content in older cultures (Fig. 2). This is in
agreement wi th the observat ions o f Mehta and Vai-
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144
P.A. Rogeret al.: Compositionof blue-green algae
dya (1978), who reported that in Nostoc cultures,
total polysaccharides, including extracellular ones,
yielded 39 of the dry ma tter in 20-day-old cultures
and 48 in 45-day-old cultures.
Major cations. Although in laboratory cultures K,
Mg, and Ca exhibited average concentrat ions of the
same order of magni tude (0 .5 ) (Table 11), very
large d i fferences were observed in so i l-grown BG A
and in natur al samples. T his was obviously due to the
high ash content in these samples, which tremen-
dously increased the Ca and Mg concentrat ions in the
harvested mater ia l.
Potassium conten t is about 1000 ppm in the soils of
the area where f ie ld samples of BG A were co l lec ted .
Therefore , average K conten t o f the ash-free a lga l
materia l collected from the field was about 0.5 .
This is similar to K content in laboratory cultures,
which indicates that K probably does not l imit BGA
growth.
Variability of the composition
An analysis of the variabil i ty of the composit ion of
labora tory cultures show ed that intragen eric variabili-
ty was sl ightly larger than intergeneric variabil i ty.
Mult ivariate analysis showed that culture age is at
least as important as the nature of the strain in
explaining the variabil i ty of the com posit ion.
Poo led da ta sh owed a ve ry large variabili ty of the
composi t ion . For example , the ra t io be tween h igher
and lower va lues recorded for N conten t was more
than 6 (Table 11). However, pooled data included
those from laborato ry cultures in which variabili ty is,
as a ru le , wider than tha t known from nature (Koma-
reck 1971): coefficients of variat ion were 31 for N
and 20 for C in laborato ry cultures, but 16 for N
and 7 for C in field samples. How ever, even when
only the data from art ificial blooms and natural
samples are consider ed, variabil i ty is sti ll high. W hen
average and e x t reme values ob ta ined for dry mat ter ,
ash, and N contents are combined, i t appears that N
conte nt in 1 tonne of fresh N2-fixing BG A averages
1.25 kg but m ay vary from 4 to 0.1 kg. Th eref ore, the
data on N2-fixing biomass in kilograms fresh weight
or d ry weight per h ectar e give li t tle informa tion on i ts
agron omic significance, which depend s mainly upon
its N content.
Implications for agronomic use of B GA in rice cultiva
tion
Total algal biomass evaluations in rice fields range
from a few kg/ha to 58 tonnes/ha fresh weight or 500
kg dry weight. Reported N2-fixing algal biomasses
range within the same l imits (Roger and Ku lasooriya
1980; Roger et al . 1985; Rog er et al. 1986). The
highest biomass currently repor ted on a fresh weight
basis in a rice field is 58 tonn es/ha for an Aphanothece
bloom in which 98.6 water and 54 ash contents
limited the N c onte nt to 13 kg/ha (R oge r et al. 1985).
The highest biomass rep orte d on a dry weight basis is
481 kg/ha , cor resp ond ing to 53 kg N/ha (Singh 1976).
Values reported by Singh indicate an N content of
11 before correct ion for the ash content . This is
very high especial ly when considering the author's
indicat ion that heavy rains frequently disturbed the
field, which implies that ash content should have
been high.
Assuming a maximum biomass of 500 kg dry
weight/ha and using average ash and N values obtain-
ed for artificial blooms and field samples (Table 11),
i t appears that the potential N contribution of an
N2-fixing bloom is 15-25 kg N/ha.
Using average values for dry matter, ash, and N
contents we are able to calculate that the average
biomass co rrespon ding to 10 kg N is about 8 tonnes
fresh weight . This is equivalent to a continuous layer
of 0.8 m m algal material o ver 1 ha of a rice field. In
other words, an algal bloom of agronomic signifi-
cance is visible to the naked eye.
Wh en looking a t BG A as a source of n i trogen for
rice the C:N rat io is a major factor determining N
mineral izat ion. E xtre me C :N values were 4.3 and 13
(Table 11). The y were observe d in laborato ry cultu-
res, which co nfirm th at variability is larger in artificial
condit ions than in nature. When data from blooms
prod uced on soil and natural samples are considered,
values range from 5.3 to 11.6 and average 8. This
indicates that BG A have a better ni trogen availabil i-
ty than organic fert i l izers such as farmyard manure
and green manures. Previous studies by Tirol et al .
(1982) showe d that availabili ty of BG A nitrogen to
two successive crops of rice was similar to that of
amm onium su l fa te.
Acknowledgements.This researchwas conductedunder a scienti-
fic agreem ent between IRR I and ORSTOM (France) and was
supported by the United Nations DevelopmentProgramme.
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P.A. Roger et al.: Composition of blue-green algae 145
Appendix
Analysis of flask cultures of N24ixing BGA isolated fro m rice fields
Ref.a Ageb Dry N C C:N Sugars Chloro- Phyeo-
no. weight phyll a cyanin
Anabaena 3 A 6.94 8,26 51,6 6.25 40.2 1.77 13.1
2 A 1.80 5.20 12.9 36.6 2.45 6.59
4 A 2.67 6.98 50.5 7.24 19,9 1.82 13.4
6 A 1.01 7.86 47.7 6.07 32.2 1.63 15.5
5 A 8.50 7.97 38.4 4.82 21.8 1.79 14.9
7 B 3.52 3.68 nd nd 27.3 0.23 6.33
4.07 6.66 51.1 7.46 30.0 1.61 11.6
Aphanothece
8 B 0.30 2.67 nd nd 76.7 0.13 1.31
Aulosira A
5.60 3.84 29.7 7.73 62.5 0.31 3.99
Calothrix
10 A 4.27 4.68 39.1 8.35 62.3 0.44 3.36
11 A 4.16 6.66 35.6 5.34 41.8 1,77 5.50
13 A 5.44 5.23 32.1 6.14 36.3 1.12 3.68
13 B 4.97 6.23 nd nd 88.1 0.32 5.65
13 B 5.33 6.03 nd nd 63.7 0.53 3.68
14 B 5.33 4.65 nd nd 41.5 0.34 2.45
14 B 5.81 4.47 nd nd 68.8 0.21 2.87
15 B 6.02 6.74 nd nd 66.9 0.46 3.16
15 B 6.03 3.70 nd nd 37,8 0.45 2.22
6.26 5.38 35.6 6.61 56.4 0.62 3.62
Fiseherella
16 A 4.91 3,40 35.2 10.34 65.5 0.33 1.96
17 A 3.47 4.99 31.9 6.39 37.0 0.58 3.45
18 A 4.41 5.26 39.7 7,55 43.5 0.93 3.07
17 B 13.1 1.78 nd nd 43.4 0.08 1.14
6.48 3.85 35.6 8.09 47.3 0.48 2.40
Gloeotrichia
19 A 4.55 4.38 42.4 9.69 38.1 1.13 3.49
20 A 2.75 5.33 36.6 6.88 49.0 1.09 5.82
20 B 0.80 2.81 nd nd 66,0 0.08 1.14
21 B 0.55 8.02 nd nd 114,1 0.42 6.58
21 B 0.80 3.95 nd nd 53.7 0.48 3,44
21 B 0,80 4.75 nd nd 64.5 0.48 3.28
1.70 4.87 39.5 8.28 64.2 0.61 3.95
Nostoc
23 A 1.11 3,52 45.8 13.0 26,6 0.81 9.25
24 A 3.95 5,12 46.8 9.14 68.4 0.34 6.08
25 A 4.44 4.00 45.1 11.3 49.2 0.56 3.79
26 A 6.33 6,89 36.9 5.36 32.0 1.72 10.4
28 A 0,28 3,71 38,2 10.2 59.2 0.63 6.72
27 A 3,34 7.15 40.4 5.65 35.4 1.35 4.91
23 B 2.59 2.97 nd nd 69.1 0.24 2.35
24 B 2.98 4.00 nd nd 48.7 0.32 5.34
22 B 1.21 5,60 nd nd 58.5 0.47 6.76
22 B 1.36 5,56 nd nd 65.9 0.44 3.74
22 B 1.36 6,17 nd nd 49.6 0.34 5.49
26 ]3 1.85 4.49 nd nd 50.6 0.21 5.68
26 B 2.95 4.20 nd nd 45.4 0.65 5.15
26 ]3 2.96 4.29 nd nd 55,1 0.44 3.73
2.62 4.83 42.2 9.12 51.0 0.61 5.67
Oscillatoria 29 A 3.32 6.42 39.0 6.07 40.0 1.05 2.80
30 B 4.00 4.37 nd nd 67.0 0.39 2.77
Scytonema
31 B 6.67 2.53 nd nd 105.1 0.12 1.29
Tolypothrix
32 A 3.12 3.87 44.5 11.5 50.5 0.86 3.32
32 B 3.64 4.99 nd nd 69.7 0.47 8.35
No, of data 46 46 22 22 46 46 46
Average 3.72 4.99 41.6 8.09 53.2 0.71 5.19
Higher value 13.1 8.26 67.3 13.2 114.1 2.45 15.5
Lower value 0.28 1.78 29.7 4.82 19.9 0.08 1.14
CV 66 31 20 31 .37 81 67
From Table 2
A: less than 4 weeks old; B: between 4 and 8 weeks old
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146 P.A. Roger et al.: Composition of blue-green algae
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