Comp Chimica

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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.



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


O n n i t r a t e 8 . 0 . . . . .


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


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 -


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



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



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



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



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 *


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 * *


* : 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



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



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



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


Aphanothece Gloeotrichia IRRI 58.8 1.75 3.82 20.4 44.5 11.6 0.074 0.388 3.29 7.20

bloom


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



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-



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



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



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-



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.



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



146 P.A. Roger et al.: Composition of blue-green algae

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Received November 27, 1985

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