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Factors affecting the production of biomass by a

nitrogen-fixing blue-green alga in outdoor culture

Article  in  Biomass · December 1987

DOI: 10.1016/0144-4565(87)90070-9

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Biomass 13 (1987) 33-43

Factors Affecting the Production of Biomass by a

Nitrogen-fixing Blue-green Alga in Outdoor Culture

Agust/n G. Fontes,* M. Angeles Vargas, Jos6 Moreno,

Miguel G. Guerrerot and Manuel Losada
Departamento de Bioqu/mica, Facultad de Biolog/a y CSIC, Apdo. 1095, 41080-Sevilla,
Spain

(Received 27 January 1987; accepted 23 April 1987)

ABSTRACT

The effectiveness of an aeration-shaking (air-lift) system for outdoor

biomass photoproduction by the N2-fixing filamentous blue-green alga
Anabaena variabilis was evaluated and the influence of relevant factors
on the productivity of the system was assessed. Air at a flow rate of 60 liters
per liter of cell suspension per h was enough, by itself, to promote
adequate turbulence and to supply the gaseous nutrients (C02 and Nz)
needed for maxirnal productivity. The addition of either or both, CO 2 and
combined nitrogen (as K N 0 3 or NH4CI), did not result in any increase in
productivity. In summer and winter, optimal cell density for a suspension
depth of 25 cm was 0"2-0"3 g (dry weight) liter -1 and 0.1-0"2 g (dry
weight) liter- 1 respectively. Reciprocally, optimal suspension depth for a
cell density of 0"2 g (dry weight) liter-1 was 20-25 cm in summer and
below 15 cm in winter. Optimal values for p H and temperature were
8.2-8"4 and 30-35°C, respectively. Under optimal conditions, mean
productivity values were about 13g (dry weight) m -2 day -1 in summer
and 6 g (dry weight) m - 2 day- 1 in winter.
Net protein content of A. variabilis cells was higher than 50%, and
nitrogen accounted for 10% of the dry biomass. From the productivity and
nitrogen content data, the N 2 fixation rate in outdoor cultures of
A. variabilis can be estimated to be higher than 1 g N m-2 day-1, i.e.
more than 3 t N per hectare per year when values are extrapolated both in
time and area;
Key words: Anabaena variabilis, algal biomass, blue-green algae,
cyanobacteria, nitrogen fixation.

*Present address: Departamento de Fisiolog/a Vegetal, Facultad de Ciencias,

Universidad de C6rdoba, Spain.
tAuthor to whom correspondence should be addressed.
33
Biomass 0144-4565/87/S03.50- © Elsevier Applied Science Publishers Ltd,
England, 1987. Printed in Great Britain
34 A.G. Fonteset al.

INTRODUCTION

The use of microorganisms for the generation of biomass with a high

protein content, suitable for different aims, is a scope of current interest.
Notwithstanding, little progress has been achieved with nitrogen-fixing
blue-green algae (cyanobacteria) in the field of biomass production as
compared with either green algae (ChloreUa, Scenedesmus) or non-N 2
fixing cyanobacteria (Spirulina). 1-3 The advantages of the N2-fixing blue-
green algae are obvious, however, since they are the only microalgae able
to fix atmospheric nitrogen and to synthesize, at the expense of sunlight,
all their cell components from water, air, and a few mineral salts, without
any requirement for combined nitrogen. These characteristics may
represent a particular economic benefit for their mass production. 4-6
A basic issue in microalgae biomass production is the accomplish-
ment of conditions under which an optimal use of incident sunlight
energy, by cells in the culture, can take place. In this context, factors such
as cell density, depth and turbulence of the suspension have a relevant
effect on productivity, provided that nutrients are not limiting and pH
and temperature are adequate. 2-7 The present study was undertaken to
determine optimal conditions for outdoor biomass production by the
N2-fixing blue-green alga Anabaena variabilis, using forced aeration in
order to provide both turbulence and a non-limiting supply of the
nutrients CO2 and N2.

MATERIALS AND METHODS

Anabaena variabilis strain ATCC 29413 (from the American Type

Culture Collection) was grown outdoors photoautotrophically at 30°C in
containers of 0.25 m 2 area and variable depth (15-55 cm). Tap-water
was employed to prepare a nitrogen-free medium, 8 but containing 4 rnM
K2HPO 4. A combined aeration-shaking (air-lift) system, which consisted
of a perforated PVC-tube frame placed on the bottom of the container,
throughout which the suspension was sparged with air or CO2-
supplemented air, was used (Fig. 1).
Elemental analysis was carried out on washed and dried samples using
an elemental analyzer, Carlo Erba Strumentazione model l106/R,
connected to a Hewlett-Packard integrator model 3390A.
Protein was estimated by the Bradford method 9 using bovine serum
albumin as a standard. Carbohydrate was analyzed by the
phenol-sulfuric acid method 1° with glucose as a standard. For lipid and
nucleic acid determinations, 8-16 ml aliquots of the cell culture were
centrifuged at 2500 g for 10 min, and the pellet was washed twice with
 Biomass from N2-fucing blue-green algae 35

1
Fig. 1. Sketch of the system used for the outdoor culture of A. variabilis, with detail of
the perforated PVC-tube frame for air sparging.

distilled water, resuspended in ethanol and maintained at 0-4°C for 1 h.

The ethanolic suspension was centrifuged as before and the supernatant
used for lipid determination according to the test for total lipids of
Boehringer-Manheim. For R N A and D N A determinations, the lipid-
free pellet was subjected to the orcinol method 11 and the diphenylamine
method,1 z respectively.
Chlorophyll a (chl) was determined following the method of
Mackinney. 13 Phycobiliproteins were estimated according to Glazer. 14
For dry weight determinations, 100-200 ml aliquots of the cell culture
were filtered through Whatman GF/C paper, washed twice, and the
filters containing the algae were dried at 80°C for 24 h. For ash
determination, about 10 nag (dry weight) samples of biomass were main-
tained at 400°C for 6 h in a muffle furnace. The heat of combustion of A.
variabilis biomass was calculated from its elemental composition accord-
ing to Minkevich and Eroshin. 15
Once a day, in the late afternoon, part of the cell suspension was
removed and replaced with fresh medium in order to maintain a
convenient cell density. The increase in biomass between two consecu-
tive dilutions was taken as a measurement of productivity.

RESULTS

Effect of air supply

A study of the effect of sparging the cell suspension with air at various
flow rates was carried out during springtime. Results in Table 1 show
36 A . G . Fontes et al.

that provision of air was growth-limiting at flow-rates of 30 liters per liter

of cell suspension per h. Productivities about 10 g (dry weight) m -2
day- 1 were achieved at a flow rate of 60 liters liter- 1 h- 1, no significant
improvement in either specific growth rate of productivity being
observed by sparging air at higher flow rates.
The sparging of air through the cell suspension at a flow rate of 60
liters liter -1 h -1 provided not only the turbulence required for a
convenient exposure to light, of the algal cells in the suspension, but also
the carbon and nitrogen required for maximal productivity. This is
substantiated by the results shown in Table 2, which clearly indicate that
neither the supplement of C O / t o the sparged air, nor the addition of

TABLE 1
Effect of A i r Flow Rate on the Productivity of A. variabilis in Outdoor Culture

Airflow rate Productivity

(liters liter- t (cell suspension) h- 9 (g (d. w.) m - 2 day- t)

0 0
30 3.9 + 0.6
60 10.1 + 1.9
90 10.9 + 2.2
120 11.6 + 2.2

Culture depth was 25 cm, and cell density was maintained at a value of 2 mg (chl) liter- t.
T h e values shown, with their corresponding standard deviations, are averages of three
independent determinations throughout three consecutive days in May.
D.w. = dry weight.

TABLE 2
Effect of Combined Nitrogen and Carbon Dioxide Supply on Biomass Productivity in
Outdoor Cultures of A. variabilis

Nitrogen source Productivity (g (d. w.) m - 2 day- I)

Air Air, 1% CO 2

N 2 (air) 13"6 + 3"2 13-2 + 3"8

KNO 3 (10 InM) 14"2 + 3"1 13"6 + 3"7
NH4CI (5 raM) 14.4 + 3"0 13"5 __.3.1

Culture depth was 25 cm, and cell density was maintained at a value of 3"5 mg (chl)
liter- 1. T h e values shown, with their corresponding standard deviations, are averages of
four independent determinations throughout four consecutive days in June.
D.w. = dry weight.
 Biomass from N2-fixing blue-green algae 37

combined nitrogen (KNO3 or NH4CI), resulted in any relevant increase

in productivity.

Effect of cell density

Cell density is a decisive parameter in determining productivity under

outdoor conditions. Its convenient modification can result in a better use
by the cells of available sunlight. Figure 2 shows the dependence of both
specific growth rate (/z) and productivity on the density of the cell
suspension in summer and winter. It can be observed that the specific
growth rate decreased with increasing cell load, even though produc-
tivity kept constant at maximal values up to cell densities of about 3.5 mg
(chl) liter -1 in summer and 1-2 mg (chl) liter -1 in winter. Maximal
productivity in summer (average irradiance 23 MJ m -2 day-]) was about
13 g (dry weight) m -2 day -], whereas in winter (average irradiance, 6-4
MJ m - 2 day- 1) was 6 g (dry weight) m - 2 day- 1.

l SUMMER WINTER ,--,

14 ½
....?
-

-o
10 ' ~

o T v
o I \o I 6 ~
¢j

o
0.1. . . . . ~ . , '\:o_; 2 ~
2 3 4 1 2 3

CELL DENSITY (mg (cht)l-')

Fig. 2. Effectof cell density on specific growth rate and biomass productivity of A.
variabilis in outdoor culture. The cultures were sparged with CO2-supplemented air
( 1:99, v/v) at a rate of 60 liters liter- l (cell suspension)h- 1.The values shown,with their
corresponding standard deviations, are averages of four and six independent
determinations throughout four and six days in December and July, respectively.

Effect of culture depth

Figure 3 shows mean productivity values in summer for cultures of

depth between 15 and 55 cm, in which minimal cell density was kept at
2 mg (chl) liter -1. Highest productivities were reached for depths
between 20 and 25 cm, with productivity values of about 13 g (dry
38 A. G. Fontes et al.

T
, Y

7E

7-o 10
>,-

>_
~ 6
o
D.
"t
is A 3'5 ' t'S SS
DEPTH (cm)

Fig. 3. Effect of culture depth on biomass productivity in outdoor cultures of A.

variabilis. The density of the cell suspensions was maintained at a value of 2 mg (chl)
liter- 1. The values shown, with their corresponding standard deviations, are averages of
four independent determinations throughout four consecutivedays in July.

weight) m-2 day-1. At depths of 15, 30 or 35 cm the productivities were

lower, ranging between 9 and 11 g (dry weight) m -2 day -1, and
decreased markedly when depth was above 35 cm.
In winter, and for a minimal cell density of 2 mg (chl) liter- l, optimal
depth was found to be about 15 cm, with a mean productivity of about
6 g (dry weight) m -2 day -1, a value which decreased markedly when
depth was more than 20 cm (data not shown).

Effect of temperature

The effect of temperature on productivity was studied in outdoor

cultures in temperature controlled cultivation systems. Productivity
increased with temperature from 15 to 35°C, the latter being the optimal
value. Above 35°C the productivity decreased markedly (Fig. 4).
In summertime, the productivity of cultures with uncontrolled
temperature was virtually the same as that of cultures maintained at
30°C, in spite of marked temperature fluctuations (20-35°C) throughout
the day in the former cultures.

Effect of pH

The influence of the pH of the medium on growth of A . variabilis within

the range 7 - 1 0 was studied. Table 3 shows mean productivity values
reached in outdoor cultures at different pH values which were achieved
by addition of bicarbonate at different concentrations. Optimal produc-
 Biomass from N2-fixing blue-green algae 39

,-.,

T
7 6 T

~ 5 Zi
~ 4
i
¢J J.

" 3
0
O.

I i I I I

15 25 35
TEMPERATURE (°C)

Fig. 4. Effect of temperature on biomass productivity in outdoor cultures of A.

variabilis. Experimental conditions were as in Fig. 2, except that the cell density was
maintained as a value of 1.5 mg (chl) liter- ~. The values shown, with their corresponding
standard deviations, are averages of four independent determinations throughout four
consecutive days in February.

TABLE 3
Effect of pH on Productivity of A. variabilis in Outdoor Cultures

NaHC0 3 added pH of medium Productivity

(mM) (g (d. w.) m- 2 day- 1)

0 7.4-7.8 7.9±2.2
5 8.2-8-4 8.5±1-8
10 9.0-9-4 4.8±0.6
20 9.7-9-9 0

Experimental conditions were as in Table 1, except that the air sparged through the
cultures (60 liters liter -l h -l) was supplemented with 0"5% (v/v) CO2. The values
shown, with their corresponding standard deviations, are averages of four independent
determinations throughout four consecutive days in April.
D.w. -- dry weight.

tivity values w e r e o b t a i n e d at p H 8.2-8.4, being slightly lower at 7 . 4 - 7 . 8

a n d d e c r e a s i n g significantly a b o v e p H 9. Actually, the cells w e r e u n a b l e
to thrive at p H 9"7-9"9.

Composition of the cells

M o l e c u l a r a n d e l e m e n t a l c o m p o s i t i o n of A . variabilis cells g r o w n
o u t d o o r s is s h o w n in Table 4. T h e cells exhibited a high p r o t e i n c o n t e n t ,
40 A. G. Fontes et al.

TABLE 4
Molecular and Elemental Composition of A. variabilisCells

Component Percentage of dry weight

Proteins 51.9 + 2.5

Carbohydrates 22.7 + 2.4
Lipids 9'2 _+0.8
Nucleic acids
DNA 1.2 ___0-2
RNA 7"5 _+1-3
Ash 4"2 + 0"9
C 49"65:0"5
O 25"9_+0"3
N 10"0+ 0"2
H 6"95:0"1

Experimental conditions were as described in Table 1, but at an air flow rate of 60 liters
liter- 1(cell suspension) h- 1.The values shown, with their corresponding standard devia-
tions, are averages of four independent determinations throughout four consecutive days
in May.

above 50% of dry weight. Interestingly, phycobiliproteins represented as

m u c h as 2 5 - 3 0 % of total net protein, thus ranging from 13 to 15% of the
dry weight. Although the chlorophyll content varied (from 0"9 to 1.6% of
the dry weight) according to cell density and depth of the cultures, it
remained fairly constant between 1.2 and 1.4% for the chosen conditions
of 3 - 4 mg (chl) liter -1 and 15-25 cm depth. Carbon, nitrogen and
hydrogen content of A. variabilis biomass were about 50%, 10% and 7%
of the dry weight respectively. From the nitrogen data, a crude protein
content (N x 6"25) higher than 60% of the dry weight could be calcu-
lated. From the elemental analysis (Table 4), the empirical formula for
the dry biomass of A. variabUis grown outdoors, can be given as
C5.8H9.702.3N, with a heat of combustion 15 of 22.6 kJ g - 1 (dry weight).

DISCUSSION

T h e results show that nitrogen fixing blue-green algae are suitable micro-
organisms for the production of protein-rich biomass when employing
sunlight as the energy source and air as the supplier of both inorganic
carbon and nitrogen, the latter being fixed at a rate of m o r e than
1 g N m - 2 d a y - 1. Maximal productivities reaching up to 17 g m - 2 day- 1
were achieved outdoors with A. variabilis in 0.25 m 2 containers using an
 Biomass from N 2-fixing blue-green algae 41

air-lift system. Experiments with 10 m 2 ponds using the same aeration

device yielded mean productivity values in summer of about 10 g (dry
weight) m - 2 d a y -1. Preliminary results using a paddle-wheel system
indicate that values over 20 g m -2 day-1 can be obtained in summer
with this blue-green alga) 7 From the values of productivity, sunlight
irradiance and heat of combustion of the algal biomass, it can be
calculated that the conversion efficiency of sunlight energy into stored
chemical energy by the system is around 2 per cent.
Productivity of outdoor microalgal cultures is largely affected by two
parameters. Firstly, the environmental factors, namely, sunlight and
temperature, are determined by the annual season and are largely
uncontrollable. Secondly, there are manageable factors, namely, turbul-
ence, cell density and suspension depth, the adequate combination of
which allows a better use by cells of incident sunlight. 3'16'18 Among the
latter factors, cell density is especially relevant, since its adequate
manipulation represents the best way of modifying the amount of light
energy available for each cell in the c u l t u r e . 16 Both in winter and in
summer, the growth rate of A. variabilis decreased in response to an
increase in cell density (Fig. 2). This is the expected performance for a
light-limited system. Maximal productivity is maintained as long as the
decrease in growth rate is compensated by the increase in cell density.
Further increase in cell density finally leads to decreased productivity.
Depth of the cell suspension is another interesting parameter; the
establishment of its optimal value being important when deciding the
design of the culture system. Our results (Fig. 3) show that, for a given
cell density, higher depth can be used in summer (23 MJ m -2 day -l
irradiance) than in winter (6 MJ m -2 day -1 irradiance), with con-
sequently greater productivity values. However, even in summer,
increasing depth culture over 20 cm has a negative effect on productivity.
Turbulence is another critical factor for productivity. In the
aeration-shaking system used in this work, the air stream promotes tur-
bulence and is the source of both gaseous nutrients (CO2 and N2). The
results indicate that a flow rate of 60 liters liter- 1 (cell suspension) h-
suffices to provide adequate turbulence, as well as all the carbon and
nitrogen needed for vigorous cell growth of A. variabilis outdoors
(Tables 1 and 2).
Optimal temperature for growth of A. variabilis is about 35°C.
Although in general the outdoor cultures have been carried out with
controlled temperature (30°C), results of experiments carried out in
summer under conditions of uncontrolled temperature, which oscillated
between 20"C and 35"C, show values for growth rate and productivity
analogous to those of temperature-controlled cultures. These results
42 A. G. Fonteset al.

indicate that A. variabilis can be grown at ambient temperature in

climatic areas like that of Seville, at least from April to October, with
substantial productivity values.

ACKNOWLEDGEMENTS

Research was supported by grants from the Comisi6on Asesora de

Investigaci6n Cientffdca y T6cnica, Fundaci6n Ram6n Areces and
Dragados y Construcciones, SA (Spain). The authors thank Mrs Antonia
Friend and Mrs M. J. P6rez de Le6n for their secretarial assistance.

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