Harvesting of Scenedesmus obliquus in
Wastewaters: Auto- or Bioflocculation?
Alain Lavoie and J. de la h u e
Dkpartement de Biolqie et Centre de Recherche en Nutrition, Universitk
Laval, Qkbec, Qukbec, Canada GtK 7P4
Accepted for publication November 26, 1986
Autoflocculation and bioflocculation are considered to be
the most promising means for the economical harvesting
of microalgae. We have therefore studied these phenom-
ena with cultures of Scenedesmus obliquus produced
during biological tertiary wastewater treatment. The
quantity of extracellular polymers produced during age-
ing of the cultures proved insufficient to initiate bio-
flocculation while the concentration of Ca2+and POa3- of
the treated effluent were t o o low t o induce auto-
flocculation. It has been shown, however, that the algae
sediment more readily upon ageing, possibly as a result
of increased cell density. The use of density gradients
made with Percoll (a colloidal solution of silica particles)
allowed measurement of the true cell density and
showed that this increases when cultures enter the de-
clining growth phase. The quality of the biomass thus
harvested is, however, considerably impaired, protein
content decreasing from 62.7% (dry wt) during the ex-
ponential growth phase (day 5) to 14% at the end of cul-
tures (day 21).
INTRODUCTION
Although chemical flocculation leads to a good har-
vesting of Scenedesmus obliquus, it would represent an
expensive step in the biological tertiary treatment of waste-
waters. Moreover, the physiological state of microalgae
plays an important part in the flocculation process. The
maximal algal recovery by chemical flocculation is obtained
at about the end of the exponential growth phase and de-
creases rapidly as soon as the cultures enter the declining
growth phase. The accumulation of extracellular polymers
at this growth stage could be acting as a protective colloid
masking the algal surface charges and leading therefore to a
reduction of flocculant efficiency.
There are only few data published on the natural floccu-
lation of microalgae. Bogan and c o - ~ o r k e r s working
,~ on
the removal of phosphorus by the filamenteous green alga
Stigeoclonium stagnatik, noted floc formation when grow-
ing this species with air bubbling. A similar phenomenon
was observed by Golueke and Oswald,s for microalgal cul-
tures photosynthetically active in a shallow pond during
warm and sunny days.
These flocculation phenomena are quite different. The
former, called autoflocculation, was first studied by
Biotechnology and Bioengineering, Vol. 30, Pp. 852-859 (1987)
0 1987 John Wiley & Sons, Inc.
Moellmer6 and more recently by Sukenik and Shelef7 who
postulated the following mechanism: increasing pH in algal
cultures, either by COPconsumption by algal photosynthesis
or by direct addition of alkali, leads the culture medium to
a supersaturation state with respect to calcium and phos-
phate ions. Such a supersaturation causes an initial nuclea-
tion of calcium phosphate precipitation which is promoted
by the algal cells serving as a solid surface. In the presence
of excess calcium ions, the calcium phosphate precipitate is
positively charged and therefore adsorbed on the negatively
charged algal cells agglomerating them and promoting algal
flocculation.
The second phenomenon, based on algal physiology, is
called bioflocculation. Two mechanisms were proposed to
explain it. Schuessler, in 1967,* suggested that biopolymers
(principally polysaccharides) produced by the algae could
be acting as a flocculant. He noted a higher production and
excretion of these products at the end of the exponential
growth phase, where maximal algal flocculability is ob-
served. Pavoni and co-workers' also showed the ability of
extracellular polymers from algal cultures to flocculate inor-
ganic colloid suspensions. The second mechanism proposed
suggested that bacteria use algal extracellular products for
growth and induce algal flocculation as they do in the acti-
vated sludges of secondary wastewater treatment. 'c-'~
Since natural flocculation can be considered as the most
promising means of economically harvesting microalgae,
we wanted to determine whether this phenomenon occurred
in Scenedesmus obliquus cultures and could be used to
harvest the algal biomasses produced during biological ter-
tiary wastewater treatment and intended for animal feeding.
Moreover, a better knowledge of the physiological reaction
of Scenedesmus obliquus is needed to enable maximal floc-
culation efficiency by natural or chemical processes as well.
MATERiALS AND METHODS
Algal Cultures
The strain of Scenedesmus obliquus used was isolated
from the secondary effluent of the Valcartier wastewater
treatment plant (Quebec, Canada). This freshwater Chloro-
CCC 0006-3592/87/070852-08$04.00
phycea, commonly found in wastewaters, is well adapted to
this environment and has been proven to be a suitable or-
ganism to perform a tertiary biological treatment of waste-
waters. l 3
Algae were grown in a lOOO-L, stainless-steel tank con-
taining 400 L secondary effluent from a wastewater treat-
ment plant located at Valcartier near Quebec. This type of
medium has the following general characteristics: N-HN,' ,
9.5 mg/L; N-N02- + N-N03-, 2.5 mg/L; P-PO.,-,
2.2 mg/L; total suspended solids, 10 mg dry wt/L; chemi-
cal oxygen demand, 30 mg 02/L; pH, 7.0. The 21-day
batch cultures were carried out at room temperature (20°C)
under 350 pE/m2/s illumination with a light: dark cycle of
14: 10. Air-bubbling (4 h-I) provided sufficient CO, for mi-
croalgal photosynthesis. Intermittent mixing (30 min every
2 h) with a propeller (?4hp motor) prevented any sedimen-
tation of algal cells. Evaporative losses were replenished
daily with distilled water. Ammonium (N-NH4+) and ortho-
phosphate (P-PO:-) ions were measured daily using a
Technicon Autoanalyzer (Industrial Methods 98-79 w/a
and 94/70 w/b, respectively) according to the APHA.14
Algal growth was monitored by transmittance at 678 nmI5
and dry weightI3 measurements.
Five different stocks of secondary effluent were used and
were identified by their sampling time: 84-06-21,84-07-19,
84-10-12, 84-12-01, and 85-01-08.
Flocculation and Sedimentation
Effect of pH
The effect of pH on algal flocculation was studied in a
standard jar test apparatus. The pH of six 1-L suspension
samples was adjusted with 1.ON NaOH or H2SO4 to values
ranging from 6.0 to 12.0; samples were then stirred at
100 rpm for 1 min, at 50 rpm for 14 min, and left still for
15 min. Samples were immediately withdrawn at the 0.75-L
level for the determination of the remaining algae after floc-
culation and sedimentation. Flocculation efficiency was es-
tablished as:
T - To
E = x 100
100 - To
where E is the percentage of algae removed from culture; T
is the algal Concentration transmittance at 678 nm after the
flocculation test; and To is the algal concentration after the
flocculation test in the control jar.
Effect of Calcium and Phosphate
The effect of calcium and phosphate on flocculation was
studied by adding KH2P04or CaC1: to obtain final concen-
trations of 0 . 2 d and 2.5mh4, respectively, in the samples,
prior to the flocculation tests.
Effect of Light and Algal Cell Density
In order to determine the effect of light on the sedimen-
tation of algae, six 1-L suspension samples were left un-
LAVOIE AND DE LA NOUE: HARVESTING OF SCENEDESMUS OBLIQUUS
agitated, three in the darkness and three under illumination
(210 pE/m2/s). After I , 2, and 4 h, the pH and the fraction
of algae still in suspension were measured in the upper-
most 250 mL supernatant of two samples (1 dark and
1 light) in the same way as for flocculation tests. All these
tests were done in the middle part of the light period of the
day-night cycle.
Cell Density
For the study of the influence of microalgal cell density
on sedimentation, cell density was measured with stepwise
density gradients of Percoll. I6 Mixing Percoll with the cul-
ture medium leads to low osmolality ( 5 2 0 mOS/kg H20)
solutions. This prevents algal cell plasmolysis and allows
the measurement of true cell density. The gradient prepara-
tion techniques have been described elsewhere.l6
Extracellular Products
The quantity of extracellular products in algal cultures
was measured according to Lewin. l7 Following two succes-
sive filtrations of a 100-mL culture sample first over a
Whatman 934 A-H then through a Millipore 0.45-pm filter,
the filtrate was concentrated to 10 mL by boiling. After
cooling, 30 mL of ethanol (95%) were added to the sample
which was then stored at 4°C overnight. The white preci-
pitate formed was centrifuged, washed with ethanol,
vacuum-dried at room temperature and weighed. Results are
expressed in milligrams of extracellular polymers per liter
of culture.
Extracellular and Capsular Carbohydrates
A 15-mL culture sample was centrifuged for 15 min at
2000g (IEC Clinical Centrifuge). The supernatant was im-
mediately used for extracellular carbohydrate measurement
by the anthrone method according to Jermyn.I8 The same
technique was used for another culture sample previously
heated at 100°C for 7 min in order to measure total carbo-
hydrate production.
The capsular fraction was the difference between extra-
cellular and total carbohydrates. Values are expressed in mg
of glucose/L.
Biomass Composition
Nitrogen, carbon and hydrogen contents of algae were
measured by combustion of lyophilised algal samples (0.2
to 1.0 mg dry wt) in a CHN analyzer (Hewlett-Packard).
Algal protein content was calculated by multiplying the
total nitrogen values by 5.6, a factor obtained from amino
acid analysis (unpublished results).
Bacterial Counts
The changes in bacterial counts were followed by the
plate count method. Starting with a dilution series of algal
853
cultures (lo-'- 0.1-mL samples were spread on nutri-
ent agar in triplicate. The agar plates were incubated for
48 h at 20°C19and colony forming units were counted.

RESULTS

Algal Culture Growth

Figure 1 shows the typical growth pattern of Scene- pH 10

desmus obliquus batch cultures. Although ammonium was

exhausted by day 6, phosphate (P-P04-3) uptake continued
up to day 9. Phosphate exhaustion coincided with the de-
clining growth phase and the stationary phase was attained
one or two days later. It can be seen, however, that the algal
biomass concentration increased continuously up to day 22.
o]
.-.
j
-.-.-.-.-.-.-._.-..o-.-.
* . , , , ,pH 7.5-8.7
Influence of pH and Culture Age on Microalgae
Flocculation 50

~-._~,._._i_._.C.-C.~.-.-.-.-.-.
The influence of the age of the culture on the floccu- loo/
0 , ,

lability of microalgae for pH values ranging from 7 to 12 is

shown in Figure 2. Without any control, the pH of the 50
cultures reached stable values comprised between 7.5 and
8.7. Effective flocculation was observed at pH 11 and 12 0
4 8 12 16 20
during the early days of culture. However, after day 6, there
Time (days)
was no flocculation at pH values between 7 and 11 and
efficiency was below 50% at a pH value of 12. Figure 2. Influence of pH and culture age on Scenedesmus obliquus
natural flocculation: (-----) onset of stationary phase (water stock:
84-06-21).

1000 1 N H ~ Phosphate ion concentration appears to be an important

0 PO, factor in the flocculation process. Figure 3 shows that
harvesting efficiency for pH value of 12 decreased as micro-
algae exhausted PO:-. Moreover, a better flocculation effi-
ciency was observed at the stationary phase for cultures
showing higher residual ortho-phosphate concentrations
[Fig. 3(A)].

1
120

80 7

40
5
%
0 , .
2
-120 3
0
Plll

-80
d
3

ln
300 - 40
ul 200
m
g 100 -0
0 4 8 12 16 20
0 2 4 6 8 10 12 14 16 18 2 0 22
Time (days)
Time (days)
Figure 3. Importance of P 0 4 3 - residual concentrations on auto-
Figure 1. Growth of Scenedesmus obliquus on wastewater (water stock: flocculation of Scenedesmus obliquus cultures at a pH value of 12, for
84-07- 19). water stocks (A) 84-06-21 and (B) 84-07-19.

854 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 30, NOVEMBER 1987

 A B
Influence of Calcium and Orthophosphate Transmittance (96)
Concentration on Scenedesmus obliquus
Autoflocculation
Figures 4(A) and 4(B) show how 2.5mM of Ca2+ or 0: , - , , , . , , , ~
J, , . , , , , , ,
0.2mM of PO:- influenced the autoflocculation efficiency Capsutar sugars (mg glucose/L)

for pH values up to 11. One can see that the addition

of calcium or phosphate significantly raised the auto-
flocculation efficiency for pH values of 10 and 11;the simul-
taneous addition of these two products allowed a good Extracellular sugar (mg glucose/L)

autoflocculation (80%)at pH values of 8 and higher. More-

over, the age of culture markedly influenced the auto-
flocculation phenomenon since the efficiency quickly
Extracellular proteins (mg/L)
decreased as soon as the culture entered the declining
growth phase (day 9), despite the addition of calcium and
phosphate.
Extracel lular polymers ( mg /L )
100-

Bioflocculation
In order to determine whether bioflocculation enables the 0; ( , , , , . , , , J, , , , , , , , , , ,
0 4 8 12 16 20 0 4 8 12- 16 20
efficient harvesting of Scenedesmus obliquus, we monitored Time (days)
the appearance of products capable of inducing bio-
Figure 5. Appearance times of products responsible for bioflocculation
flocculation during algal culture. Figure 5 shows that the induction in Scenedesrnus obliquus cultures: ( .1 ) onset of stationary phase
quantities of extracellular polymers varied during the course for water stocks (A) 84-10-12 and (B) 84-12-01.
of incubation and did not appear to be correlated with the
age of the cultures. The polymer concentrations measured in
different cultures never exceeded 120 mg/L, even if the carbohydrate and nonprotein material. The amount of ex-
values obtained include considerable quantities of non- tracellular proteins, variable for different cultures, was
comprised between 0 and 7 mg/L in all the experiments.
Capsular and extracellular sugars appeared at the onset of
A B
the declining growth phase.
While the amount of extracellular sugar showed few varia-
tions during the cultures, that of capsular material increased
0 4 , . . . , 4 , . , , , , ,~
continually until the end of culture. The maximal concen-
100 trations obtained during this study were 5.2 mg/L for extra-
cellular and 25.4 mg/L for capsular sugars.

Microalgal Sedimentation
The efficiency of sedimentation of Scenedesmus obliquus
was closely related to the physiological state of the algae as
shown in Figure 6. Although maximal efficiency varied be-
tween cultures, observed tendencies were consistent, sedi-
mentation increasing with declining growth phase. One can
also see that light influenced this phenomenon. When cul-
tures were kept under darkness, more algae sedimented and
more rapidly. Although illumination caused the pH to rise to
100
10, no floc formation was observed under these conditions.
?=-\ h
pM PO:

’J ‘0, 1
.r-.-.-...--.-.+, .‘ I-.-.-.-.‘
I Cell Density
Higher microalgal sedimentation with culture ageing is
4 8 12 16 20 4 6 8 10 12
not related to flocculation phenomena (no floc formation);
Time (days)
we therefore followed the progression of cellular density
Figure 4. Importance of calcium and phosphate for autoflocculation of during two batch cultures in order to find out whether this
Scenedesrnus obliquus and the influence of culture age on this phenome-
non: ( 1) onset of stationary phase for water stocks (A) 84-10-12 and (B)
parameter could explain microalgal behavior. Figure 7
84-12-01; (0)control; addition of (0)Ca2+; (M) PO.,-,(0) Caz+ and shows the distribution of algae in different density classes
Po:-. and their relative abundance in each of these classes for the

LAVOIE AND DE LA NOUE: HARVESTING OF SCENEDESMUS OBLIQUUS 855

 --
30 30 25 25 5 5 5 5 5 5
60 60 60 60 15 15 10 15 10 10 15
10 10 15 1 5 80 4 0 25 25 25 25 20 25 5 5 5 5 5
4 0 25 15 20 25 35 35 30 3 0 3 0 30 2 0 20
35 15 15 20 20 30 30 3 0 35 35 40 40
25 25 15 5 5 15 15 10 10 15 10
5 112
0
5 5 5 5
15 15
5 5
15 15
5 1 0
15 20

T
t
15 10
17 7 0 60 10 20 20
66 15 30 34 35 40 25 20 15 5 5 5 5 5
17 66 20 10 20 40 40 30 30 25 25 25 20 20 10

0 I Y 35 20 10 2 0 25 30
10 20 10 S 15
5 5 5 10
3 0 3 0 30
15 20 15
10 10 10
30 40 50 4 0
15 10 5
10 10 5
10
10
8t 1'2 1'6 20 4 8t 1'2 1'6 20 5 10 10 10 10 15 15 20 2 0 30
. . .
4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
Time (days)
Time (days)
Figore 6. Effect of culture ageing on algal sedimentation in (A) darkness
and (B) under illumination. Sedimentation duration was (0)1 h, (0) 2 h, Figure 7. Relationship between the density of Scenedesmus obliquus
and (m) 4 h. Water stocks were 84-12-01 (bottom) and 85-01-08 (top). The cells (their distribution (%) in different density classes) and sedimentation
arrow ( T ) denotes the onset of declining growth phase. efficiency in darkness (expressed as algal recovery (%)). Sedimentation
duration: 4 hours. Water stocks were (A) 84-12-01 and (B) 85-01-08.The
arrow ( $ ) denotes the end of the exponential growth phase.

entire duration of 21-day cultures. One can see that
cell densities showed lowest values when algae were experi-
encing exponential growth phase and that the algal popu-
lation was distributed into two or three density classes
(1.090 g/mL or less). As the cultures aged, classes of higher
density appeared and algal populations became more and
more diversified (from four to eight density classes).
Sedimentation under illumination being always less effi-
cient than under dark conditions, despite identical initial cell
density in both cases, density measurements were made with
the cells remaining in suspension under both conditions.
Table I shows that decantation in the dark allowed a marked
sedimentation of algae with density higher 1.090 g/mL
while illumination prevented such a selective sedimentation.
Biomass Composition
Table I1 summarizes the variations in chemical com-
position of two experimental populations of Scenedesmus
obliquus. One can see that the protein content of microalgae
dropped quickly at the end of exponential growth phase. The
carbon content of biomass decreased lightly in the early
stationary phase, but gradually rose afterwards. It can also
be noted that the hydrogen content increased during station-
ary phase.
Bacterial Counts
The typical changes in bacterial counts during S . obliquus
batch culture on secondary effluent are illustrated in

Table I. Influence of light on the sedimentation of Scenedesmus obliquus: distribution of algae (%)
in different density classes.

Density (g/mL)

~~

Culture A 17.5% 35% 17.5% 10% 10% 10%

Supernatant L" 12.5% 37.5% 37.5% 12.5% - -
Supernatant Db 100% - - - - -
Culture B 25% 33% 18% 8% 8% 8%
Supernatant L 11% 33% 33% - 11% 11%
Supematant D 100% - - - - -
Culture C 25% 33% 25% 8.5% - 8.5%
Supernatant L 11% 33% 33% - 11% 11%
Supernatant D 50% 50% - - - -

"This is the algal distribution in the supernatant after a 4-h sedimentation under illumination.
'This is the algal distribution in the supernatant after a 4-h sedimentation in darkness.

856 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 30, NOVEMBER 1987

 Table 11. Influence of culture age on chemical composition of Scenedesmus obliquus

Age Percent C Percent H Percent N Percent protein

(days) @') (%I (%)

7 53.9 9.7 9.2 51.2

11 46.6 5.8 4.2 23.6
15 46.7 7.4 3.4 19.0
19 49.5 22.5 3.0 16.8
End of exponential growth D-7; beginning of stdonary phase D-10.
5 52.5 7.5 11.2 62.7
8 51.7 8.8 9.9 55.4
14 46.3 14.8 4.3 24.1
22 53.0 24.0 2.5 14.0
End of exponential growth D-8; beginning of stationary phase D-l 1.

Figure 8. The number of bacteria clearly decreased as the
algal population was growing exponentially and reached lo3
colony forming units/mL, a low value subsequently main-
tained.
DISCUSSION
Autoflocculation
The autoflocculation phenomenon has been usually asso-
ciated with intense photosynthetic activity. The resulting
alkaline conditions induce a coprecipitation of magne-
sium, calcium, phosphate, and carbonate with algal cells."
Sukenik and Shelef7 identified Ca2+and PO-: as the main
causes of this phenomenon, the respective concentration of
these ions determining the pH value required to induce auto-
flocculation.
The poor recovery of algae obtained during our experi-
ments with pH values lower than 12 (Fig. 2) suggests that
one of these ions, if not both, was limiting. Moreover, it can
be seen from Figure 3 that flocculation at pH 12 was closely
related to the residual phosphate concentration in the culture
medium. However, the PO:- concentrations measured on
days 5 and 6 could allow autoflocculation induction at pH
values higher than 9.3 provided that sufficient Ca2+concen-
trations were present.' Nevertheless, no flocculation was
observed for pH values lower than 11 while the maximal
160
10,
: I \
.-
5 10 15 20
Time (days)
Figure 8. Changes in bacterial population during Scenedesmus ob[iquus
culture. Water stock was 84-10-12.
efficiency obtained at this pH was below 45%. The low
calcium content of our culture medium (6 mg/L) explains
such a result. In fact, a minimal calcium concentration of
40 mg/L is reported as necessary for maximal auto-
flocculation.' This implies that calcium addition would be
required to induce autoflocculationin most of the secondary
effluents as the calcium content of freshwaters is
ca. 25 mg/L.21 In warm climates, evaporation can be high
enough to increase the calcium concentration, hence the
induction of autoflocculation.5-7
The simultaneous addition of 2 . 5 d calcium and 0.2mM
orthosphosphate allowed autoflocculation induction from
pH 8.0 with an improved and constant efficiency for pH 9,
10, and 11 [Figs. 4(A) and 4(B)]. These results agree with
those of Sukenik and Shelef; this was expected since we
used the ion concentrations proposed by these authors to
allow maximal autoflocculation at pH values higher than
8.5. The separate addition of CaZ+or POP- allows identi-
fication of the limiting ion. An example of calcium limi-
tation is shown in Figure 4(A). One can see that the addition
of Ca" alone, while culture phosphate concentration was
still considerable (D-5), caused a significant improvement
of flocculation efficiency at a pH value of 9. Nevertheless,
from day 8, while orthophosphate concentration was re-
duced, the simple addition of Ca2+was insufficientto induce
algal flocculation under the same pH condition. An inverse
relationship can be seen for a culture that presented low
phosphate concentrations. It can be seen in Figure 4(B) that
the simultaneous addition of calcium and phosphate was
rewired in order to obtain a significant flocculation at pH 9,
both. ions being limiting. Moreover, adding Ca2+ alone
was insufficient, even at pH 10, while adding orthophos-
phate was sufficient to maximize flocculation under this pH
condition.
Even in the presence of Ca2+and PO:- concentrations
capable of maximal autoflocculation induction (pH > 8.0),
the percentage of algae recovered by sedimentationdropped
abruptly as soon as the cultures entered the declining growth
phase. This behavior is similar to that observed during stud-
ies on chemical flocculation of microalgae'~~ and shows the
importance of algal cell physiology for the flocculation
process.

LAVOIE AND DE LA NOUE: HARVESTING OF SCENEDESMUS OBLIQUUS 857

Bioflocculation
The results obtained during the study of the influence of
pH on microalgal flocculation (Fig. 2) as well as the con-
centrations of extracellular sugars and polymers found in the
cultures (Fig. 5 ) show that Scenedesmus obliquus har-
vesting could not be effected by bioflocculation, under our
culture conditions (batch cultures using secondary effluent
with excess C02provided through air bubbling). Since the
pH of our cultures did not exceed values of 7.5-8.7, we can
infer from the work of de la Noue et a1.22that the availability
of CO, was sufficient under the present conditions. In fact,
according to Pavoni and co-workers,' bioflocculation occurs
when pH is higher than 10 and its efficiency improves with
culture ageing when the lysis of old cells contributes by
increasing the amount of extracellular polymers responsible
for floc formation.
Our results show that no significant algal flocculation
could be induced after the eleventh day of culture whatever
the medium pH (Fig. 2). Moreover, the higher extracellular
polymer concentration measured after 2 1 days of culture
never exceeded 120 mg/L for all our experiments, a value
markedly inferior to that of 500 mg/L reported by Pavoni
and co-workers.'
Although massive production of extracellular poly-
saccharides by any microalga is possible, blue-green and
filamenteous species are the most suitable organisms.23
Comparing our results with those of Pavoni and co-workers'
is not an easy task, since they did not identify or describe the
algae they used. These authors concluded that extracellular
polymers are responsible for bioflocculation by comparing
their results to those they obtained previously for bacterial
population^,^^ without considering algal autoflocculationor
sedimentation.
Capsular and extracellular sugars appeared at the onset of
the declining growth phase for Scenedesmus obliquus.
These results agree with those obtained by Samson25who
studied the physiology of Oocystis sp., another fresh-
water Chlorophycea. This author reported that Oocystis
grown on synthetic medium excretes proteins only a few
days after the beginning of the stationary phase. The pres-
ence of extracellular proteins in our cultures from the very
beginning, is explained by the use of secondary effluent as
culture medium.
The appearance of capsular sugars coincided with the loss
of flocculation efficiency reported in Figures 4(A) and 4(B)
as previously reported during chemical flocculation stud-
ies.' The protection of algal surface charges by bio-
polymers, as proposed by Tenney et a1.,3 could therefore
have been the principal cause for the slight flocculability of
Scenedesmus obliquus on attaining the stationary phase.
The low bacterial population observed in Scenedesmus
obliquus batch cultures, grown on secondary effluent in
the laboratory, eliminated the possibility of alga-bacteria
co-flocculation as reported by Eisenberg et al." with
Ankistrodesmus sp. grown on raw sewage. These authors,
studying bioflocculation of microalgae in oxidation ponds,
noted that flocs were composed of algal cells incorporated
into the matrices of filamentous bacteria.
858 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 30, NOVEMBER 1987
Sedimentation and Cell Density
Although the results obtained point out that Scenedesmus
obliquus did not experience the required conditions for bio-
flocculation under our culture conditions, the sedimentation
of algal cells appeared to be closely related to their physio-
logical state (Fig. 6). In fact, the percentage of algal re-
covery was lowest during the exponential growth phase,
increased rapidly during the declining growth phase and
remained about constant at the stationary phase.
The variation of sedimentation in the dark (duration of
4 h) followed a similar pattern to that of cell density class
modifications (Fig. 7). The increase in recovery efficiency
was coincident with the appearance of algal cells with a
density higher than 1.090 g/mL, when they entered the
declining growth phase. The highest recovery efficiency
attained was observed when there was no longer any density
class lower than 1.090 g/mL. This factor, however, should
not play a major role in harvesting systems for larger scale
operation due to the importance of other factors and to
differences in culture modes.
The nonselective sedimentation observed under illu-
mination (Table I) was responsible for the lower algal recov-
ery (Fig. 6 ) and can be explained by two factors. The first
one, entirely physical, is the induction of thermal con-
vection currents. The second one, purely physiological, is
oxygen production via algal photosynthesis. Oxygen micro-
bubbles appears on the algal cell surfaces and makes the
algae float.
Algal Chemical Composition
The rapid decrease of algal protein content when cultures
entered the declining growth phase is common for Chloro-
phy~eae.,~ The consumption of intracellular stocks of nitro-
gen and protein excretion are two mechanisms that can
explain this phenomenon.
The hydrogen content increase that accompanied the car-
bon content restoration is indicative of the accumulation of
photosynthetic products ,26 mainly saccharides, that could
have been responsible for the increase in cell density.''
Work is under progress to clarify this point.
CONCLUSIONS
The amounts of extracellular polymers produced during
the ageing of cultures as well as the low Ca2+ and PO:-
concentrations in the secondary effluents used appear to
be insufficient to allow bio- and autoflocculation respec-
tively of Scenedesmus obliquus under our laboratory con-
ditions. The exhaustion of phosphate is in any event one
of the main purposes of biological tertiary treatment, and
the use of calcium as a flocculant gives harvested biomass
of insufficient quality for feeding purposes. Consequently,
autoflocculation does not appear a suitable process for har-
vesting Scenedesmus obliquus if biomasses are intended for
feeding purposes.
Although the algal sedimentation without flocculation im-
proves with the ageing of cultures, this harvesting process
remains unstable and very slow. Moreover, the protein con-
tent of algae decreases quickly as soon as the cultures enter
the declining growth phase. If the harvested biomass must
be of a fair quality for animal feeding purposes, chemical
flocculation, especially with chitosan as reported else-
where,2 presently appears to be the best way to harvest
Scenedesmus obliquus cultures at least until new polymeric
flocculants are tested and proved to be safe for such a pur-
pose. However, the results obtained indicate the possibility
of a significant reduction of the flocculating agent concen-
tration by acting before the microalgae enter the declining
growth phase, i.e., before the appearance of capsular sugars
and at a time when the protein content of the biomass is
maximal.
This work was supported by grants from the Natural Sciences and
Engineering Research Council of Canada (NSERC) and the Fonds
F.C.A.R. from the Ministry of Education of QuCbec (team grant).
We wish to thank C. Dallaire for the drawings, D. Ni Eidhin for
help in the writing of the final version, and G. Gagnon for typing
the manuscript.
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