BIOTECHNOLOGY AND BIOENGINEERING

VOL. XVI, PAGES 881-896 (1974)

Growth of Spirulina maxima Algae in Effluents

fkom Secondary Waste-Water Treatment Plants

N. KOSARIC, H. T. NGUYEN, and M. A. BERGOUGNOU,

Chemical and Biochemical Engineering Department,
The University of Western Ontario, London, Ontario

Summary
S p h l i n a maxima, a high protein alga, was grown in effluents from the London
municipal waste treatment plant. Optimum growth conditions were developed,
the composition of algae and the removal of nitrogen and phosphorus in effluents
were studied. The advantages of this process in tertiary waste-water treatment
and the quality of the single cell protein were investigated.

INTRODUCTION
Spirulina maxima is a blue-green alga belonging to the family of
oscillatoriaceae. It is a large-size microscopic, multicellular alga
which grows in shallow ponds of high salinity and alkalinity in tropi-
cal countries (Fig. 1).
S. maxima has been used as food since ancient times in some re-
gions of Africa and Mexico. Nutritional studies, carried out by the
Institut FranGais du Petrole (IFP) showed that Spirulina is one of
the richest protein sources ever found,' the protein also contains all
essential amino acids in a proportion comparable to other conven-
tional protein foods and satisfies the composition recommended by
FA0 except for methionine.2 S. maxima as a food source also sup-
plies a considerable amount of fat, carbohydrate, vitamins, and
Tests carried out on rats and chickens, showed that
Spirulina has a high nutritive value and a good dige~tibility.~
Considering the nutritional values of S. maxima and the relative
simplicity in cultivation and harvesting of this alga because of its
large size compared to other microalgae, several pilot plant studies
have been performed in France, Algeria, and Mexico to produce
S. maxima. The algae were cultivated in synthetic media made up
mainly of aqueous mineral salt solutions and a carbon source from
881
@ 1974 by John Wiley d Sons, Inc.
882 KOSARIC, NGUYEN, AND BERGOUGNOU

combustion gases containing carbon dioxide. The cost of chemicals

was estimated t o be about 15% of the total production cost.2 There-
fore a more economical way t o culture S. maxima would be desirable.
From another aspect, one of the major problems in the pollution
of inland waters today is the excessive enrichment of waters by nu-
trients, especially nitrogen and phosphorus compounds, which are
capable of supporting growth of undesirable algae blooms. These
blooms, upon dying, cause taste and odor problems in lakes or rivers.
Nost of the nutrients come from industrial or domestic sewage since
the conventional treatment processes are relatively ineffective in re-
moving these nutrients. Normally, only 20 t o 40% of nutrients are
removed from sewages unless special procedures are adopted for re-
moval. One of the possible methods for removing large amounts of
nutrients from waste waters is through biological removal using algal
culture.
I n this study, the algae Spirulina maxima were grown in effluents
from the secondary municipal waste-water treatment plant in Lon-
don, Ontario. Factors influencing the growth and values of the
process with regard to nutrition and tertiary Waste-water treatment
were investigated. The objectives were twofold: a) t o treat the
effluents for nutrient removal (i.e., nitrogen and phosphorus) and b)
to produce economically a biomass with good nutritional character-
istics, suitable for animal or human consumption.

MATERIALS AND METHODS

Materials
The original culture of S. maxima was supplied on agar slants from
Plant Physiology Institute, University of Gottingen, Germany.
The waste-water effluent samples were obtained from the Green-
way Pollution Control Centre, which receives and treats domestic
and industrial sewages in the London community. The samples were
collected a t the effluent site of the final settling tank in the activated
sludge process.
Algal Growth Systems
System 1
The algae \yere grown in 250 ml Erlenmeyer flasks, each containing
100 ml of medium. The cultures were continuously illuminated at
4OOO lux using 40-watt fluorescent tubes. The experiments were
 GROWTH OF SPIRULINA IS WASTE WATERS 883

Fig. 1. Microscopic photograph of S. maxima (magnification X200).

performed in the “walk-in” incubator where the temperature was

kept constant a t 30 f 0.5”C.
Air was bubbled through the culture medium a t the rate of 2 w m .
Additional C 0 2 ,adjusted to 2% by volume in air, was passed through
intermittently to act as carbon source and to maintain the pH of the
cultures in the range of 8.5 to 10. Experimental cultures were in-
oculated from developing stock cultures grown under the same
conditions.

System 2
Further experiments have been carried out in a larger scale culture
container; a 5-gal bottle containing 10 liters of culture medium.
The culture was illuminated by four “Gro-Lux” fluorescent tubes.
The aeration and mixing of the cultures were accomplished by air
containing C 0 2 which was passed into the bottle at the rate of 0.5
w m and by a magnetic stirrer. The incubation temperature was
maintained at 30 2°C. The pH was controlled in the range of
8.5 to 10 by adjusting the COZ content in air. Approximately 0.5 g
dry weight of inoculum was added to the medium and the sample
was collected through a sample tube.

System 3
For open culture of algae, a small-scale pond has been constructed
and studied. A schematic of this is presented in Figure 2.
684 KOSARIC, NGUYEN, AND BERGOUGNOU

SEPARATING /%

D I R E C T I O ~ J OF
CULTURE FLOW

- AIR A N D C 0 2
ENTRANCE

Fig. 2. Scheme of the culture pond.

The pond, made of Plexiglas, was a shallow tank measuring 120

cm x 40 cm x 7 cm and supplied with wells at each end. It was
separated into two compartments by a wall which had an opening at
the bottom of each well t o allow intercommunication between the
compartments. Air, supplemented by COz, was injected into the
well in one of the two compartments a t the rate of 5 liters/min.
The bubbles of air then decreased the average density of the fluid in
the compartment and the heavier liquid in the adjacent compartment
then flowed in and set up a circulation effect. This “air-lift” tech-
nique thus provided both the circulation of the culture needed for
efficient photosynthesis and COz for algal growth.
The capacity and the culture surface area of the pond was 26.90
liters and 4680 cm3, respectively. The thickness of the suspension
was always maintained at 4 cm by continuous addition of water t o
supplement the loss due to evaporation.
The addition of the culture medium during the semicontinuous
study was performed by a sigmamotor pump. The harvesting of
S. maxima was relatively simple since the algae, when matured,
float on the surface in large clumps which may easily be skimmed
off from the surface.
Analytical Met hods
Analyses of waste-water samples
The total nitrogen content of waste waters was determined by the
Coleman 29A Nitrogen Analyzer.
 GROWTH OF SPIRULINA IS WASTE WATERS 885

The total phosphorus in the water samples was first converted to

orthophosphate by acid hydrolysis. The orthophosphate content
was then determined using the vanadomolybdophosphoric acid colori-
metric method.6
Analyses of biomass
The algal biomass was collected by filtration and i t was washed
several times by distilled water to remove dissolved salts. The
clean samples were then either dried to constant weight at 105"C,
when only the dry weight determination was required, or freeze-dried
and stored at 0°C in the refrigerator for chemical analysis.
The moisture and ash contents in freeze-dried samples were deter-
mined by the method described by Jacobs.?
The nitrogen content of the freeze-dried S . maxima was determined
by the Coleman model 29A Nitrogen Analyzer. Protein was as-
sumed t o contain 16% nitrogen and consequently, a factor of 6.25
was used to convert nitrogen value t o the protein content in the
sample.
For amino acid determination, the dry sample was hydrolyzed by
refluxing with 5.7N HCI in nitrogen atmosphere for 24 hr. The
hydrolyzate was evaporated to dryness under vacuum. Norleucine
was then added as internal standard and the aliquot was analyzed
by an automatic Technicon TSM-1 Amino Acid Analyzer.
The carbohydrate content was determined using anthrone reagent.&
The absorption was measured a t 540 nm using a Spectronic 20
Colorimeter.
The fat content in the algal sample was extracted by chloroform
and chloroform-methanol and determined gra~imetrically.~

FACTORS INFLUENCING GROWTH

In order to obtain the information on the growth characteristics
of S. maxima in the secondary effluents, the important growth
parameters were investigated using growth system 1.
Spirulina maxima has been known t o grow and live under alkaline
conditions, thus an inadequate pH could easily inhibit the growth.
The sensitivity of S. maxima to pH is illustrated in Figure 3 where
the algae were cultivated in synthetic medium under controlled pH;
high yields were obtained in a pH range of 9 t o 10, the optimum pH
being around 9.5. Growth was drastically reduced a t pH 11 and
completely inhibited at pH 8 or lower. Since the initial pH of the
Greenway secondary effluents range between 6.8 t o 7.3, usually the
886 KOSARIC, NGUYEN, A N D BERGOUGNOU

Fig. 3. Effect of pH 011 yield of S. maxima after 12 days of culture in synthetic

medium.

initial pH must be adjusted t o about 9.5 by addition of 1 g/liter of

sodium bicarbonate, which also acts as buffer in the waste-water
medium to prevent a rapid deviation of pH during growth.
Typical growth curves of S. maxima in synthetic medium and
Greenway effluent are shown in Figure 4. The growth was per-
formed under constant illumination of 4,000 lux a t a temperature of
30 f 0.5"C and pH of 8.5 to 10. The growth in both media tended
t o proceed exponentially in the first few days and the growth rate
increased with cell concentration. The growth in synthetic medium
then increased linearly and optimum cell concentration was reached
after 14 days. The growth rate in Greenway effluent reached the
optimum at about 6 days and decreased afterward; the maximum
cell concentration of 0.77 g/liter was attained after 9 days of growth.
The growth rate as well as the yield in Greenway effluent was lower
 GROWTH OF SPIIIULINA I N WASTE WATERS 887

DAYS OF CULTURE

Fig. 4. Growth curves of S. maxima i l l 250 ml Erlenmeyer flasks at 30T,

4,000 lux and aeration of 2 liter/liter/min.

than that in synthetic medium which may be caused by nutrient

deficiency in the waste water.
The effect of illumination on the growth is shown in Figure 5.
A higher light intensity clearly improved the growth in every phase
as it increased the photosynthesis and cell activity.
The effect of temperature on growth was studied in the range of 10
t o 45°C (Fig. 6). The optimum temperature for growth was between
30 t o 40°C in the synthetic medium and between 25 to 35°C in the
Greenway effluent. The growth was completely inhibited a t 10°C
and 45°C. The results showed that S. maxima are thermophilic
algae. However, the algae could not live long a t high temperatures.
At 40"C, S. maxima only grew actively in the first 4 days and then
started t o die.
The duration of illumination also affected the growth of S. maxima.
The growth rate as well as the yield are reduced by dark periods.
The growth curves of S. maxima under continuous culture and 12 hr
intermittent illumination are compared in Figure 7. The effect of
diurnal intermittent illumination was relatively less serious in Green-
way effluent than in synthetic medium.

CHEMICAL COMPOSITION OF Spirulina maxima

The algal biomass was recovered by filtration, washed several
times with distilled water, and then freeze-dried. The freeze-dried
samples were analyzed for protein,. carbohydrates, fat, ash, and
888 KOSARIC, NGUYEN, AN11 BERGOUGNOU

1
5.500 lux

2.0 &
o.../ lUl

c
-
M

1.2 -
s GREENWAY E F F L U E N l
I*
0 -

Fig. 5. Effect of light intensity 011 the growth of S. maxima.

moisture contents. The spectra of amino acids were also determined

to establish the value of the protein fraction in the algal cells.
The analytical results of the algal biomass grown in Greenway
effluent were compared to that grown in synthetic medium under the
same growth conditions (Table I). The protein content of X. maxima
grown in synthetic medium was 63.1%, it is comparable to the results
reported by thc IFP (62 to 6S70). The algae grown in Greenway
effluent showed a difference in characteristics depending on the time
of growth. The analysis of algal samples after 6 days of batch cul-
ture in Greenway effluent showed that the protein content decreased
slightly from 63.1 to 50.5% and the fat and carbohydrate contents
increasd. The effect was more serious after 10 days on the sta-
 GROWTH OF SPIRULINA IK WASTE WATERS 889

2.0 -
SYNTHETIC MEDIUM
-
1.6 -
-

1.2 -

’
0 1*2[ GREENWAY EFFLUENT

I I I I I
0’ I I
0 4 8 12 16
DAYS

Fig. 6. Effect of temperature on the growth of S. maxima.

tionary phase of growth where the protein content of the algal bio-
mass was only 28.3% and the fat and carbohydrate contents increased
as high as 9.2% and 43.2%, respectively. The ash and moisture
contents were relatively unchanged.
A possible explanation of the variation in chemical composition
of the algae is the nutrient deficiency in Greenway effluent during
the batch culture. When the limited amount of nutrients in the
medium has been used up by the algae during the active growth
phase, a decrease in protein was observed. As a consequence, a
higher amount of carbohydrate and fat was accumulated in the algal
cells during the later phase of growth.
890 KOSARIC, NGUYEN, AND BER.GOUGNOU

-1
-1
w
CJ
0.5

0 I I I I I I I
0 4 8 12 16
DAYS

Fig. 7. Effect of diurnal intermittent illumination on the growth of S. muzi72iu.

TABLE I
Chemical Composition of S. maxima Biomass (% by wt)

Synthetic Greenway Effluent

Medium 6days 10 days

Protein 63.1 50.5 28.3

Carbohydrate 16.6 26.5 43.2
Fat 1 .o 4.6 9.2
Ash 13.9 14.0 14.5
Moisture 5.0 4.8 4.7
 GROWTH OF SPIRULINA IN WASTE WATERS 89 I

This phenomenon has also been reported by several authors.

Lewin'O noted a decrease of nitrogen content from 8-10% of the dry
weight t o 2Y0 and a n increase of fat content to a value as high as
80% in several species of Chlorella and Scendedesmus algae during
the nitrogen deficiency periods. According to Durant and Jolly,ll
by appropriately modifying the culture conditions, the producer can
vary the concentration of protein from 7 t o 85%, that of fat, from 4
to 8670, and that of carbohydrate from 5 to 38% in Chlorella. This
response has been considered as an attractive property by Russian
scientists since the composition of algae could theoretically be ad-
justed t o suit the nutritional needs of cosmonauts when the algal
systems are used in space programs.12
Amino acid spectra were also taken for the biomass samples
grown in synthetic medium and Greenway effluent. Despite the
protein content deviation, the composition of amino acids in the
8. maxima biomass is similar in different media (Table 11). All
essential amino acids are found in the biomass and their proportion is
comparable to the F A 0 standard. The only limiting amino acid is
methionine, which is the case for almost all single cell proteins.
According to these findings, an excellent nutritional quality of the
protein is expected. However, no further nutritional studies to es-
tablish biological and toxicological values of this protein were per-
formed.
TABLE I1
Essential Amino Acids in S. maxima (g per 100 g protein)

FA0 Synthetic Greenway Effluent

Amino Acids Combination Medium 6 days 10 days

Isoleucine 4.2 5.80 5.87 5.71

Leucine 4.8 9.31 9.60 9.26
Lysine 4.2 5.00 5.43 4.68
Phenylalanine 2.8 4.03 4.44 4.45
Methionine 2.2 2.28 2.12 2.10
Threonine 2.8 4.99 4.81 5.35
Tryptophan 1.4 a B a

Valine 4.2 6.92 7.55 6.62

a Not reported

REMOVAL OF NITROGEN AND PHOSPHORUS IN

WASTE-WATER EFFLUENTS
The waste-water samples of the present study were collected from
the local waste treatment plant. The characteristics of the effluents
vary according t o the time of sample collection. Usually, the efflu-
892 KOSARIC, NGUYEN, AX11 BERGOUGNOU

ents contain about 10 to 30 ppm of &day biological oxygen demand

(BOD), 28.2 to 52 ppm nitrogen, and 0 to 4.8 ppm phosphorus.
The effect of S. maxima growth on the removal of nitrogen and
phosphorus was studied in both batch and semicontinuous culture.
The batch culture was performed in the growth system 2. The
removal of nitrogen and phosphorus was studied in the Greenway
effluent containing initially 40.8 ppm nitrogen and 4.8 ppm phos-
phorus. The inoculum was about 0.5 g of cell dry weight in 10 liters
of culture medium. The nitrogen and phosphorus content of the
Greenway effluent was reduced simultaneously during the growth of
S. maxima. The nitrogen content was reduced almost 100% after
8 days of growth (Fig. 8), the phosphorus content was about 1.73
ppm a t this time (64% removal). The removal of nitrogen was
relatively unchanged with excess phosphorus content, the average
removal rate was still about 5 mg/liter/day at the initial phosphorus
concentration of 86.4 ppm. When the initial nitrogen concentration
was increased to 179 ppm by addition of potassium nitrate, the nitro-
gen content decreased t o 51 ppm after 10 days of growth while the
phosphorus content in the effluent decreased continuously Trom 4.8
t o 0.83 ppm or 82.7%.
During semicontinuous culture of S. maxima in the culture pond
(growth system 3), the water effluents were collected and analyzed
for nitrogen and phosphorus. The results are illustrated in Figure 9.
With the initial concentration of nitrogen and phosphorus of 52 ppm
and 4.2 ppm, respectively, and at a detention time of 4 days, the
mean removal of nitrogen was 87% and the removal of phosphorus
was calculated to be about 60%.
Thus, during both batch and semicontinuous cultures, the removal
of nitrogen is much faster than that of phosphorus. Nitrogen, rather
than phosphorus, tends t o be the limiting factor in Spirulina culture
even if the initial N : P ratio in the Greenway effluent was 10:1 or
higher. This phenomenon may be explained by the high content of
nitrogen in the algal biomass, all nitrogen compounds in the waste
water are assimilated by the algae, which utilize light energy, carbon
dioxide, and inorganic constituents to produce energy containing cell
material. A small part of nitrogen may be lost to the atmosphere
in the gaseous form of ammonia because of the alkaline condition in
the Spirulina culture.
The phosphorus compounds are taken up in a relatively smaller
amount. Since the algae contain about 10% nitrogen and only 0.5
t o 1% phosphorus, the removal rate is consequently much higher for
nitrogen. The phosphorus uptake is also dependent on the metabolic
 G R O W T H OF SPIKULINA I?; WASTE WATERS 893

Fig. of

activity of algae, the uptake amount is negligible when the algae are
at the stationary phase and higher during the active growth phase.
I n gencral, the removal of nutrients from waste water depends on
many factors, such as the amount of algae in the culture, the initial
concentration of nutrients, and the culture conditions.

PRELIMINARY DESIGN OF AN ALGAE PLANT

Information gained from the present investigation, together with
t h a t from studies of S. maxima culture in synthetic media carried
out by the IFPISmake i t possible to combine the treatment of waste
894 KOSAHIC, NGUYEN, AND BERGOUGNOU

-&
c

.6 - P

si
0 -4-/ /-
V
-1 .2-
-1
W
0 - I I I I I I I

and algal production where S. maxima is cultivated in waste-water

effluents and C02-containing waste gases.
The proposed process flowsheet is presented in Figure 10. Munici-
pal or industrial sewage is first introduced into a primary settling
tank where the majority of suspended solids are removed by sedi-
mentation. The sludge from this settling is further treated in an
anaerobic digester. The digestion by-product, methane, is collected
and used as additional fuel in the power plant. A segregate COZ
GROWTH OF SPIRULINA I S WASTE WATERS 895

L
896 KOSARIC, NGUYEN, AND BERGOUGNOU

gas stream released from the digester and formed by the combustion
of methane is passed to the algal pond and the produced power is
used in the plant operations. The excess flue gases are sent to the
stack.
The effluent from the primary settling tank then enters the aera-
tion tank where the organic matter in the sewage is metabolized by
microorganisms with a consequent BOD reduction. The resulting
sludge is then settled in the final settling tank. The clarified effluent
from this tank, almost free of BOD and containing nitrogen and phos-
phorus, is fed continuously to the culture pond where algae grow in
sunlight. The algal product is harvested, dried, and heat sterilized
before i t is ready for human or animal consumption. A detailed
design of the plant is in progress.
This work waa supported by the University of Western Ontario Research
Council. Analytical assistance from Labatt’s Breweries Ltd. is appreciated.

References
1. S. A. Miller in Single Cell Protein, R. S. Mateles and S. R. Tannenbaum,
Eds., M.I.T. Press, Cambridge, Mass., 1968.
2. C. Meyer, C. R . Seance Etude SOC.Fran. Thermiciens, Paris, 1969.
3. F. Busson, Spirulina platensis (Gom.) Geitler et Spirulina Geitleri. J . de
Toni Cyanophycees Alimentaires, Service de sante, France, 1970.
4 . H. Nakamura, Rep. from the Spirulina Development Committee of
Japan, 1970.
li.G. Clement, C. Riddey, and R. Menzi, J . Sci. Food Agr., 18, 498 (1967).
6. Standard Methods for the Examination of Water and Wastewater, 13th ed.,
.4mer. Pub. Health Assoc., Washington, D.C., 1971.
7. JI. B. Jacobs, The Chemical Analysis of Food and Food Products, 3rd ed.,
D. Van Xostrand Co., Princeton, N. J., 1965.
8. A. C. Seish, Rep. No. 46-83, 2nd Revision, Nat. Res. Council of Canada,
1952.
9. N. Kosaric, Ph.D. Thesis, The University of Western Ontario, Canada,
1969.
10. R. A. Lewin, Physiology and Biochemistry of Algae, Academic Press,
New York, 1962.
1 1 . N. W. Durant and C. Jolly, Fish. Znd. Res., 5, 67 (1969).
12. P. A. Lachance in Single Cell Protein, R. S. Mateles and S. R. Tannenbaum,
Eds., M.I.T. Press, Cambridge, Mass., 1968.

-4ccepted for Publication December 24, 1973