Biomass l 1 ( 1986) 61-74

P r o d u c t i o n of Spirulina Biomass in Closed

Photobioreactors

G. Torzillo, B. Pushparaj, F. Bocci, W. Balloni, R. Materassi

and G. Florenzano

Centro di Studio dei Microrganismi Autotrofi del CNR e Istituto di Microbiologia

Agraria e Tecnica dell'Universit~ di Firenze, Piazzale delle Cascine 27, 1-50144,
Firenze, Italy

(Received 20 June 1986; accepted 1 August 1986)

ABSTRACT

The results of a six year investigation on the outdoor mass culture of

Spirulina platensis and S. maxima in closed tubular photobioreactors are
reported. On average, under the climatic conditions of central Italy, the
annual yield of biomass obtained from the closed culture units was
equivalent to 33 t dry weight ha -1 year-k In the same climatic conditions
the yield of the same organisms grown in open ponds was about 18 t ha -I
year -1. This considerable difference is due primarily to better temperature
conditions in the closed culture system. The main problems encountered
relate to the control of temperature and oxygen concentration in the
culture suspension. This will require an appropriate design and manage-
ment of the photobioreactor as well as the selection of strains specifically
adapted to grow at high temperature and high oxygen concentration.

Key words: Spirulina, outdoor mass culture, tubular reactor, photobio-

reactor.

INTRODUCTION

In recent years several research groups have investigated the growth of

different photosynthetic m i c r o o r g a n i s m s in tubular systems. Pirt et al.
have developed the t h e o r y and design of a tubular photobioreactor.
T h e y have c o n s i d e r e d s o m e i m p o r t a n t parameters, such as turbulent
flow of the culture, e n e r g y r e q u i r e m e n t for culture circulation, 02 and
c a r b o n dioxide d e m a n d and supply. 1 With a p h o t o b i o r e a c t o r m a d e of
61
Biomass 0144-4565/86/S03.50- © Elsevier Applied Science Publishers Ltd,
England, 1986. Printed in Great Britain
62 G. Torzillo et al.

very narrow tubes (1 cm bore size) they attained very high photosyn-
thetic efficiency of fight energy conversion into algal biomass. 2 Gudin
and coworkers have investigated the growth of microalgae in closed
culture devices of up to 100 m 2 with the aim of producing valuable extra-
cellular products, such as polysaccharides from Porphyridium spp. and
Chlamydomonas mexicana or hydrocarbons from Botryococcus spp. The
problems investigated by Gudin include: thermal regulation of the
culture, choice of material and culture management. 3,4
Tubular photobioreactors have several advantages over conventional
open ponds: they can be erected over any open space, can operate at
high biomass concentration and keep out atmosphere contaminants.
Moreover, the process can be optimized by fermentation control
principles and computer application, 5 so as to achieve the maximum
utilization of solar energy at all times. Finally, loss of water by evapora-
tion is eliminated. However, photobioreactors function like solar collec-
tors in that they can reach high temperatures to adversely affect the
growth of the majority of photosynthetic microorganisms. Culturing of
typical mesophilic strains requires the installation of expensive cooling
facilities; but the problem of temperature control becomes less critical if
more thermotolerant organisms are grown. Spirulina platensis and
Spirulina maxima can tolerate temperatures as high as 40°C for a few
hours without appreciable adverse effects. For this reason the authors'
Research Centre started an investigation several years ago on the use of
tubular photobioreactors in the outdoor mass culture of Spirulina spp.
The research was aimed at the optimization of biomass yield and its
biochemical composition. This report illustrates the characteristics of the
experimental culture equipment and the main results obtained. The
performance of the tubular culture device is compared with that of open
ponds.

MATERIALS AND METHODS

General description of culture equipment

The pilot plant for growing Spirufina and the equipment for circulating
the culture suspension and harvesting the biomass are shown in Figs 1
and 2. The photobioreactors are made with transparent tubes. At the
beginning flexible polyethylene tubes (14 cm diameter and 0-3 mm
thickness) were used (Fig. 3). Owing to their inadequate mechanical
strength, they were replaced by plexiglass tubes having 13 cm inner
diameter and 4 mm thickness. These diameters were selected to achieve
 Production of Spirulina biomass in closed photobioreactors 63

.... ..... : :i:!~:~ii;!

iiiiiii!ii~'i!i~l'i li!iii i!

Fig. 1. General view of the tubular pilot plant. Tubes made of polymethyl methacrylate
(plexiglass).

Fig. 2. Details of the ancillary equipments of the closed culture plant showing the
harvesting and circulation systems.

a surface to volume ratio similar to that of open ponds (about 100 litres
of culture suspension per m 2 of illuminated surface). Each photobio-
reactor is made up of several tubes laid side by side on a white poly-
ethylene sheet and joined by PVC connections to form a loop. Each
connection incorporates a narrow tube for oxygen degassing. At the exit
64 G. Torzillo et al.

Fig. 3. Viewof the photobioreactormadeof polyethylenetubes.

of the tubular circuit the culture suspension falls into a receiving tank. A
diaphragm pump (model MA 180, AGI POMPE, Milan) raises the
culture to a feeding tank containing a siphon that allows an intermittent
discharge into the photobioreactor. At intervals of 4 min about 350 litres
of culture suspension are discharged into the photobioreactor, thus
moving the culture in the tubes at a rate of 0.26 m s-1. This regime of
circulation is obtained by adjusting the flow rate of the pump to 4000
litres h-1. The result was better than continuous circulation at the same
flow rate of the pump. The maximal length of the circuit was 500 m,
corresponding to a volume of 8000 litres and a surface of 80 m 2. The
surface area of the photobioreactors was calculated on the basis of the
surface area effectively occupied by the tubes plus the interspace
between them (about 3 cm).
The temperature of the culture suspension inside the tubular circuit is
monitored with several thermistor probes (LSI model TT-3) appro-
priately positioned. The partial 02 pressure in the culture is determined
polarographically with two Clark-type electrodes placed at the inlet of
the culture in the photobioreactor and its outlet. The amount of solar
radiation has been measured with a solarimeter MICROS equipped with
a pyranometric sensor (Kipp and Zonen Type CM 5/6).

Organisms and culture conditions

Spirulina maxima strain 4MX and Spirulina platensis strain M2 of the

culture collection of the authors' Research Centre were used. The
 Production of Spirulina biomass in closed photobioreactors 65

culture medium has been described elsewhere. 6 It contains 24 g litre- ~of

sodium bicarbonate + carbonate. The pH was maintained to 9.4-9.8 by
CO2 addition. The temperature control of the culture suspension inside
the photobioreactor was achieved in different ways (see results). The
culture operated with a semicontinuous regimen. The amount of biomass
harvested each day was adjusted in order to achieve a biomass concen-
tration of 0.6 g dry weight litre- ~or 1.2 g dry weight litre-1, correspond-
ing to an areal density of 60 and 120 g m -2 of illuminated area,
respectively. During harvesting operations the culture suspension was
withdrawn at a constant rate for a time corresponding to that required by
the culture to cover the whole tubular circuit. The biomass was
harvested by filtration on a vibroscreen with a net of 50 ktm pore size.
The culture solution was recycled into the photobioreactor after the
necessary additions of nutrients. The harvested biomass was washed
twice with deionized water and sundried. Once a week a sample of
biomass was analysed for nitrogen content. The concentration of
nitrogen and phosphorus in the culture medium was checked once a
week. The amino acid pattern and fatty acid composition were deter-
mined occasionally on representative samples of the biomass.

Analytical procedures
Biomass concentration in the culture was determined daily. The cells
from a representative sample were filtered, washed with deionized water
and dried at 105°C for 3 h before weighing. Total nitrogen content of the
biomass was determined with an automatic nitrogen analyser (ANA
1500, Carlo Erba Strumentazione, Milan). Fats were extracted with
chloroform-methanol (2:1 v/v) for 24 h in a Soxlet extractor, dried at
75°C for 1 h and weighed. In order to determine the fatty acids composi-
tion, the lipid extract was dissolved in a methanol: H2SOa:benzene
mixture (97.9:2:0-1% v/v), placed in a flame-closed vial and heated for
4 h at 75°C. The methyl esters of fatty acids were extracted with ether,
washed with salt water and concentrated to a small volume for gas
chromatographic analysis. GL analysis was performed with a
Perkin-Elmer Sigma 2 gas chromatograph with a Supelco SP 2330 60 m
capillary fused silica column and a FID detector. The temperature of the
injector was 250°C and the oven was programmed from an isotherm of
130°C for 6°C rain-1 to a final temperature of 210°C with a rate of 3°C
rain- 1. The carrier gas was He, at a pressure of 30 psi and the split was
80:1. The retention times of the peaks were compared with those of pure
standards and the quantitative estimation was performed with the aid of
a Perkin-Elmer LCI 100 integrator. The amino acid pattern was deter-
66 G. Torzilloet al.
mined with an automatic aminoacid analyser (Model 3A27 Carlo Erba
Strumentazione, Milan). Phycocyanin content was estimated colorimetri-
cally after extraction in phosphate buffer (pH 7), applying the extinction
coefficient of O'hEocha. 7

RESULTS

Temperature control of the culture

Since a large amount of heat is generated inside a photobioreactor

exposed to natural sunlight, in the absence of an effective dissipation
mechanism, a considerable increase in the temperature of the culture
suspension can occur. This system is advantageous in temperate condi-
tions but becomes problematic in warm climates. In Florence we
observed that the temperature inside the photobioreactor on a sunny day
reaches values 10-13°C higher than the air temperature for 2-3 h.
Hence during summer months temperatures higher than 40°C were very
common. Since our strains of Spirulina maxima fail to grow above 36°C
and are killed by exposure to temperatures higher than 42°C for 2-3 h
over 2-3 consecutive days, it was necessary to devise a cooling system.
Three systems of cooling were tested:
(i) Shading of the tubes with dark-coloured plastic sheets. For an effec-
tive control of the temperature it was necessary to cover about 80% of
the surface for 5-6 h daily. This caused a strong reduction in the amount
of solar radiation received by the culture and consequently in the yield of
biomass.
(ii) Overlapping two or three tubes. This system was difficult to instal
and inadequate for effective control of the temperature inside the
photobioreactor.
(iii) Cooling the culture by spraying water on the surface. The system
was operated when the temperature of the culture reached a critical
value. For Spirufina maxima 4MX, whose maximum temperature for
growth was 36°C, water spraying was started at 35°C and stopped at
33°C. In the climatic conditions of Florence the cooling device worked
about 40 days year- 1. The amount of water lost by evaporation ranged
from 1 to 2 litres m -2 day-1. This cooling system functioned very
efficiently. However, in order to reduce the amount of water required for
cooling, from 1983 a thermotolerant strain of Spirulina was utilized:
Spirulina platensis M2. This organism was able to grow up to 42°C and
could tolerate a daily exposure up to 46°C for at least 3 h without being
killed. Cooling the circuit to avoid temperatures higher than 44°C
 Production of Spirulina biomass in closed photobioreactors 67

proved adequate for attaining a high yield of biomass. In the last three
years the cooling systems worked about 20 days year-1. The amount of
water sprayed was less than 250 litres m-2 year- J. Only a fraction of this
water was lost by evaporation.

Influence of the circuit length on productivity and on protein content of

the biomass

The length of the tubular circuit exerts a considerable influence on the

oxygen concentration in the culture suspension. After the entry of the
culture into the photobioreactor, the photosynthetic activity of the
biomass causes an increase in the oxygen concentration in the liquid at a
rate that depends on light intensity and temperature. For a given tem-
perature an increase in light intensity causes an increase in the rate of
oxygen evolution by the culture. Similarly, for a given light intensity an
increase of temperature induces an increase in oxygen production, at
least as long as the temperature remains below the optimum values for
growth (Table 1). Obviously, the oxygen concentration in the culture
suspension increases as the culture moves forward in the tubular circuit.
On a summer day the oxygen concentration in the culture reaches
20-25 ppm one hour after entry of the culture into the photobioreactor.
W h e n the culture comes out from the tubular circuit a certain amount of
oxygen is lost in the atmosphere. The action of the circulation pump is
very important in 02 degassing. In Fig. 4 the yields of two photobio-
reactors of 250 and 500 m length are shown. In the former the culture
takes 2 h to cover the whole circuit length while in the latter it takes 4 h.

TABLE 1
Influence of Temperature and Light Intensity on the
Increase in O2 Concentration (mg 02 litre 1) in a
Culture of Spirulina' maxima 4MX After One Hour in
the Tubular Photobioreactor

Light intensity Temperature C C)

(BE m -2 s-l)
16-20 21-25 26-30

200 0"9 1'3 2"6

200-400 2-9 3"7 4'8
400-?00 3-0 4-3 5'2
"/00-1000 5-0 6-3 7-4
1000-1400 -- 6-6 8"0
1400-1800 -- -- 8"8
68 G. Torzillo et al.

2s
E

m
E
o

"; 5
>, i i i I i

M J J A S
months

Fig. 4. Yield of Spirulina maxima 4MX grown in tubular photobioreactors of different

length (as specified in the Figure) circulated at the same speed.

TABLE 2
Biochemical Composition of Spirufina maxima 4 M X
Grown in Photobioreactors of Different Length (% of
Dry Weight)

Length of
photobioreactor

250 m 500 m

Crude protein 62.0 55-5

Total lipids 13-3 13.2
Carbohydrates 18-0 22.6
Ash 6.7 8"7

The short photobioreactor gave higher yields. The biomass produced in

the long photobioreactor had a lower protein content, counterbalanced
by a higher carbohydrate content (Table 2). Taking into account that no
significant differences in the temperature profile or in biomass concen-
trations occurred in the two photobioreactors, the influence of the length
of the tubular circuit on the yield of biomass and on its biochemical
composition must be attributed mostly to the adverse effect of high
oxygen concentration on protein synthesis in Spirulina as described
recently by the authors. 8

Influence of cellular concentration on the yield and composition of

biomass

The output of biomass of the photobioreactors was inversely related to

the concentration of biomass in the culture. The yield of biomass
 Production of S pirulina biomass in closed photobioreactors 69

obtained in various years from cultures maintained at 0.6 g and 1.2 g dry
weight litre-~ are reshown in Fig 5. In general the cultures run at 0"6 g
litre -1 were more productive. The differences in yield were more
marked in July (about 35%) and declined in autumn, probably as a
consequence of the limiting effect exerted by low temperature. Biomass
concentrations below 0.6 g litre -1 are not practicable because the
cyanobacterial cells are damaged by the combined effect of high light
intensity and oxygen concentration. The biomass concentration of the
cultures influences the phycocyanin level. In August the phycocyanin
content in the culture at a concentration of 1"2 g litre-~ was 25% higher
than in the culture at 0.6 g litre -l. This difference increased to 40% at
the beginning of October, while at the end of October the phycocyanin
content became equal. A decrease in total nitrogen content of the
biomass occurred at the beginning of August (Fig. 6). The change was
more marked in the culture run at low cellular concentration and
coincided with a sudden lowering of cellular concentration due to a
higher filtration rate of the cultures. After three days the normal cellular
concentration was restored and the nitrogen content returned to the
previous values. The protein content of the biomass (about 54%) was
relatively stable, irrespective of the cellular concentration of the culture.
In August the nitrogen content of biomass was higher in the culture
grown at lower cellular concentration. Presumably in this culture the
nucleic acid content was higher due to the higher growth rate reached by
the cyanobacterial population. No significant differences in the fatty
acids composition and in the amino acid pattern of the biomass grown at
low and high cellular concentrations were found.

~ 0,6 g t "+
I 1,2 g t -1

N
~.

months

Fig. 5. Influence of cellular concentration on the yield of Spirulina platensis M2 grown

in a tubular photobioreactor.
70 G. Torzillo et al.

• 1.2 gl1
N*/,
o 0.6 gl -I
13

11

10

i i i

Ju(y August September

Fig. 6. Nitrogen content of Spirulina platensis M2 grown at different cellular concen-
trations.

photobioreacfor
25

:,.. 5

A M J J A 5 0

20 4 ~g
o
10 2 .~_ *'<
r~

A M J J A S 0
months

Fig. 7. Comparison of the mean yield of Spirulina platensis M2 cultivated in photo-

bioreactors and in open ponds.

Comparative yield of both open ponds and photobioreactors

In Fig. 7 the course of productivity of Spirulina platensis M2 cultivated in

photobioreactors and open ponds operating at the same areal density is
shown. The daily yield of the photobioreactor was higher than that of the
open ponds during the whole cultivation period. This was mainly due to
better temperature profiles in the closed culture system. The culture in
the photobioreactors reaches optimum temperature earlier in the
 Production of Spirulina biomass in closed photobioreactors 71
o
16 September
~0 photobioreacfor

w 20
m i 1 1 I i

;Q. oC photobioreacfor
E t 25 July. ~

2o
i i I

5.00 8.00 1100 lt, O0 17.00 hours

Fig. 8. Typicalcourseof the temperatureof Spirulina cultures in photobioreactorsand

ponds in July and September.The arrow indicatesthe start of the cooling.

morning and better temperatures for the growth are also reached during
the months in which the cultivation in ponds is limited by low tempera-
tures (Fig. 8). No significant differences in the protein content and amino
acid composition between the biomass grown in photobioreactors and in
open ponds were found (Table 3). However, some differences were
found in the fatty acids composition. Biomass grown in open ponds
showed a higher degree of unsaturation of fatty acids due mainly to a
higher content in 7-linolenic acid (Table 4). It is reasonable to suppose
that these differences are dependent on the higher temperatures reached
in the photobioreactor.

DISCUSSION

The results obtained from the investigations, extended for several years,
on the growth of Spirulina platensis and S. maxima in a tubular photo-
bioreactor at a pilot plant scale, allow a preliminary evaluation of the
merits and drawbacks of closed culture systems, in comparison with
open ponds, for mass culturing of oxygenic phototrophic microbes.
Under the climatic conditions of central Italy, the average yearly
output of biomass from the photobioreactor was nearly 90% higher than
that obtained from open ponds. This difference is due primarily to the
fact that closed photobioreactors allow a considerable extension of the
cultivation period (215 days against 175 days with open ponds), because
72 G. Torzillo et al.

TABLE 3
Amino acid Composition of Spirulina platensis M2 Grown in Tubular
Photobioreactor and in Open Pond (g amino acids 100 g protein- ~)

Amino a c i d s Photobioreactor Open pond

1. Aspartic acid 11.2 11.4

2. Threonine 5.1 4.5
3. Serine 4.3 3.7
4. Glutamic acid 17"4 16.9
5. Proline 4.4 4.4
6. Glycine 5"6 5.6
7. Alanine 7'2 7.4
8. Cystine 0'3 0"3
9. Valine 7.9 8.1
10. Methionine 2.2 2"4
11. Isoleucine 6.9 7.1
12. Leucine 10.5 10"6
13. Tyrosine 4.7 4.8
14. Phenylalanine 5'1 5.4
15. Lysine 5"8 5"9
16. Histidine 2"1 2.1
17. Ammonia 1"1 1.1
18. Arginine 8"3 8'3
19. a-e-Diamino pimelic acid 1.0 1.0
Protein % 53.6 51.0

TABLE 4
Main Differences in Fatty Acids Composition of Spirulina platensis M2 Grown in
Tubular Photobioreactor and in Open Pond (% of total fatty acids)

CI6:0 CI6: l A 9 C18: I A 9 C18:2A 9,12 C18:3A 6, 9,12

Photobioreactor 52.3 4.2 3.8 23.0 13"8

Open pond 48.0 6"5 3"0 21'5 18"1

inside the closed culture units the d a y t e m p e r a t u r e attains values con-

siderably higher t h a n a m b i e n t temperature. Additionally, the tubular
culture device gave slightly better yield also in the w a r m e s t p e r i o d of the
year, w h e n air t e m p e r a t u r e s reach 3 0 - 3 5 ° C . Also in this p e r i o d a better
t e m p e r a t u r e profile in the p h o t o b i o r e a c t o r , due to a m o r e rapid heating
 Production of Spirulina biomass in closed photobioreactors 73

of the culture in the early morning, presumably plays a major role in

determining the better yields observed.
T h e main problems encountered in the operation of tubular photo-
bioreactors are the control of oxygen concentration and of overheating
in summer. T h e problem of temperature control has been greatly
alleviated with the selection of a strain of Spirulina platensis able to grow
up to 42°C and to withstand temperatures of 46°C for a few hours
without adverse effect. With this strain cooling of the photobioreactor by
spraying water on the culture tubes was required only for 20 days year-
and the amount of water required was estimated to be 250 litres m-2 of
cultivated area year-1. T h e control of oxygen concentration requires an
appropriate design of the photobioreactors, so that the culture can be
degassed before the oxygen tension in the culture suspension becomes
harmful. The selection of a Spirulina strain less sensitive to the adverse
effect of high O2 tension would contribute significantly to simplifying the
construction of large photobioreactors as well as their management.
Although the installation of large scale photobioreactors may be more
costly than a production plant based on open ponds, the greater
efficiency in solar energy conversion that can be achieved with closed
systems makes them very attractive in photosynthetic biomass produc-
tion through mass culture of microalgae and cyanobacteria.

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