algal autofiocculation verification
TRANSCRIPT
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Algal Autof Ioc cu lation Ver i f icat io n
and Proposed Mechanism
A. Sukenik and
G.
Shelef
Sherman Research Center Technion
Israel Institute of Technology Haifa 32000 Israel
Accepted for publication Ju ly 12 1983
Biomass autofloccula tion in outdoor algal cultures was
found to be associated with increases of culture pH
levels, due to CO2 consumption by the algal photosyn-
thetic activity. Under these alkaline conditions, some
medium chemical ions precipitated together with the al-
gal biomass. The chemical substances involved with the
process and it s dependence on pH value were studied by
simulation of autoflocculation in laboratory experi-
ments. Proper concentrat ions of calcium and orthophos-
phate ions in the medium are important for autofloccula-
tion and, in order to a ttain it with in the pH range 8.5-9.0
the culture should contain 0.lmM-0.2mM orthophos-
phate and 1.5mM-2.5mM calcium prior
to
raising the pH
level. Calcium phosphate precipitates are considered as
the flocculating agent which reacts with the negatively
charged surface of the algae and promotes aggregation
and flocculation.
INTRODUCTION
The mass culture of microalgae can be practiced to
attain different objectives, including algal protein pro-
duction, production of various organic substances, waste-
water treatment, solar energy conversion, or combina-
tions of these objectives. Almost all of these uses of mass
algae cultures should include the separation of the micro-
algae from the aqueous medium. However, because of
their small size (5-50 pm) and negative surface electric
charge, microalgae form stable suspensions and are dif-
ficult to separate and recover.2
The apparent spontaneous floc formation and settling
of microalgae and bacteria in algal ponds or high-rate
algal ponds has been mentioned in the literature for two
decade^.^ The phenomenon was termed autofloccula-
tion. In most cases, this phenomenon was associated
with elevated pH due to photosynthetic C 0 2consumption
corresponding with precipitation of magnesium, calcium,
phosphate, and carbonate salts with algal cells.4 Aside
from this coprecipitative autoflocculation, the formation
of algal cell aggregates can also be due to:
1)
excreted or-
ganic macromolecule^,^ 2) inhibited release of microalgae
daughter cells,5 and
3
aggregation between microalgae
and bacteria.6 In countries with ample sunshine, auto-
flocculation can be brought about by proper adjustment
Biotechnology and Bioengineering,
Vol. XXVI,
Pp.
142-147 (1984)
of environmental conditions in a settling pond. This prin-
ciple has found technical application for removing micro-
bial biomass from treated ~ as t e w at e r . ~he method is not
sufficiently reliable at present, however, and the under-
lying mechanism has hitherto remained ~ n c l e a r . ~
The objective of this work is to elucidate coprecipitative
autoflocculation under alkaline conditions. The physio-
logical activity of the algal biomass may change the cul-
ture pH level in different directions. Photosynthesis ni-
trate and phosphate assimilation increase the pH level
while respiration and ammonia assimilation decrease
iL8g9The intensity and the rate of these changes depend on
the medium buffer capacity and the rate of the biological
reactions. The most important reaction which may in-
crease the pH of an algal culture is photosynthesis since
nitrogen and phosphorus are assimilated at least 6.5 and
100 times, respectively, less than carbon. Under such al-
kaline conditions, several inorganic salts may precipitate
thus affecting the stability of the algal suspension and
cause flocculation. Autoflocculation phenomenon, asso-
ciated with raised pH due to photosynthesis activity, is
quantitatively verified in this study and the mechanism for
microalgal autoflocculation is proposed based on labora-
tory and outdoor experiments.
MATERIALS AND METH OD
Culture Conditions
Outdoor Scenedesmus dimorphus (TURP.) K U T Z cul-
tures were grown in 120-L model ponds, 35 cm deep,
stirred by rotating paddle armes.
A
modified medium, en-
riched with 2.25mM CaCI2 and 0.6mM MgS04 was used.
Salts were dissolved in tap water or a combination of tap
and distilled water to reduce the alkalinity. Carbon diox-
ide was added to the culture by sparging pure pressurized
COz through a pH-activated solenoid valve as a function
of photosynthetic activity. lo Cultures were maintained for
various growth periods under batch conditions with a
full supply of C 02 and continuous agitation. Following
the growth period, autoflocculation was initiated by
1984
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stopping the COz supply and agitation for a sedimenta-
tion period.
In parallel with the above outdoor experiments, indoor
cultures of
Sc. dimorphus
and of
Chlorella vulgaris
BEIJ
were grown in Bolds medium (Table I) in 4-L culture
tubes at room temperature. Continuous illumination was
provided by fluorescent lamps.
A t
the end of the expo-
nential growth, cells were harvested by centrifugation,
washed twice and resuspended in Bolds basal medium
whose calcium magnesium and phosphate concentrations
were modified. These suspensions were used
in
jar tests
which simulated algal autoflocculation.
Flocculation Tests
The effect of pH on algal flocculation was studied in a
standard jar test apparatus. The pH of each 1-L suspen-
sion sample was adjusted with NaOH
(0.1N)
o preplanned
value, whereupon six samples were stirred at first at 80
rpm for 1 min, followed by
30
rpm stirring for 15min, and
then left unagitated for
15
min. Immediately thereafter
samples were withdrawn at the
0.8-L
level for chemical
analysis and determination of algae residues after floccu-
lation and sedimentation.
Analytical Method
Algal biomass was determined gravimetrically while the
orthophosphate concentration was determined by vana-
domolybdate reaction. Magnesium and calcium concen-
trations were measured in a Perkin-Elmer model 460
atomic absorption spectrophotometer.
Since algal biomass concentration was found to be cor-
related linearly with the suspension optical absorbance at
420 nm, algal growth and algae residues after flocculation
and sedimentation were assessed by optical absorbance
measurements. The percentage of algae removed by floc-
culation and sedimentation in the flocculation tests were
determined according to
x
100
o c
P = -
LO
where P is the percentage of algae removed from the cul-
ture;
C
is the algae concentration after the flocculation
test (mg/L); and C is the algae concentration after the
flocculation test in the control jar (mg/L).
Table
I.
according to
ref.
1 1
Bolds basal medium composition. Trace-elements were added
Concentration
Salt mg/L)
NaNO,
250
CaC12.2Hz0 25
MgSO . 7H2O 75
K2HP04 75
KH2PO4
17.5
NaCl 25
The algal surface electric charge was assessed by mea-
suring the velocity and direction of cell migration in a
known electric field, hence electrophoretic mobility, using
a zeta meter. Deriving the zeta potential Z p ) f a particle
from its electrophoretic mobility is shown by
zp=
44?F
Em
D
where Em is the electrophoretic mobility (cm2/V/s); 7 is
the viscosity (g/cm/s); and
D
is the dielectic constant
(C/V/cm).
The zeta potential is the only measurable potential in
the vicinity of a colloidal particle and is directly related to
the particle surface potential. The surface electric charge
of a particle is evaluated from the particle surface poten-
tial and its capacity.*
RESULTS
AND DISCUSSION
Algae Autoflocculation in Outdoor Pond
Batch cultures of
Sc.
dimorphus
were grown in model
outdoor ponds under field conditions using modified me-
dium. A typical autoflocculation experiment is shown in
Figure 1. Algal batch culture was grown autotrophically
for seven days while culture pH was kept constant at 7.0 by
the controlled addition of C 0 2 according to the culture
photosynthetic demand.
On
day eight autoflocculation
was initiated by stopping pond agitation as well as the car-
bon dioxide supply. As shown in Figure
1,
this autofloc-
culation initiation coincided with a rapid increase
in
pH
up to
9.0
due to COz consumption by the photosynthetic
activity of the culture. During this pH rise, algae were
observed to aggregate into fragile flocs and sedimented to
the ponds bottom leaving low concentration of algae in
suspension as evident by the absorbance levels of less
than 0.05 at the ninth day. The consequential changes in
the chemical composition of the culture medium 24 h af-
ter the autoflocculation initiation is given in Table 11.
The reduction in orthophosphate, calcium, and alkalin-
ity concentrations indicate chemical precipitation to-
gether with the algal biomass. These changes are typical
for alkaline conditions and their effect on algal autofloc-
-
0.51
9.0
8.05
7.0
N
-
5:
a
a 0.1
2 4 6 8 1 0
9.0
8.05
7.0
b
8 0.3
e -
5:
a 0.1
N
m
a
2 4 6 8 i o
time
(d)
Figure 1 Autoflocculation in an autotrophic outdoor culture of Sc.
dimorphus . T h e ar row indicates autoflocculation initiation by ceasing
pond
agitation and CO, supply.
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Table II.
autoflocculation initiation.
The changes in outdoor Sc.
dimorphus
culture 24 h after
At autofloc culation 24 h after
Param eter initiation in it iation
PH 7.2 8.9
Absorbance at 420 nm
0.55
0.02
Total suspen ded solids
Orthophosphate
mg/L)
310 10
mg/L
as
P) 4.8 0.2
Calcium mg/L) 160 152
Magnesium mg/L) 42 42
Alkalinity
d)
2.0
1.6
culation is described later in this paper. The biomass re-
maining in suspension was low as evident by the reduc-
tion of total suspended solids down to
10
mg/L; this
corresponds to an algae removal efficiency of
96 .
Effect
of Light
In order to determine the effect of photosynthetic activ-
ity
on
algae autoflocculation, cultures were grown auto-
trophically for six days.
On
the seventh day, autofloccula-
tion was initiated as described above and
ai
that point
some of the cultures were covered to prevent light penetra-
tion and to stop photosynthetic activity while other cul-
tures continued to be exposed to solar irradiance (ca. 4.5
E/m2 h). Optical absorbance profiles (correspondent to
algal biomass concentration) of the light-exposed and the
covered ponds are shown in Figure 2. In the light-exposed
cultures, biomass concentrations decreased rapidly with
time. Two hours after autoflocculation initiation, 30
mg/L biomass was measured in the upper level of the
pond while four hours later the whole pond depth was
almost free of algae biomass (Fig. 2). The pH in the illumi-
nated cultures rose rapidly and, four hours after autofloc-
culation initiation, pH 8.9 was measured in the entire
culture depth. The algae biomass concentration in the
covered cultures decreased at a considerably slower rate
than in the illuminated ones. Two hours after autofloccu-
lation was initiated, 150 mg/L of algal biomass was mea-
L i g h t E x p o s e d C o r e r a d
0 2 0.4
0.6
3 0
0.2
0 . 4
0.6
A b s o r b o n c e
1420
n m ) Ab r o r b o n c o
I 4 2 0
n m )
ire
2
Optical absorbance profiles of light-exposed and covered
ponds
0 )
h ,
m) h ,
and
(A)
h aft er autoflocculation initiation. Ini-
tial algal biomass concentration 220 mg /L.
sured in the upper level of the pond. Four hours later, 35
mg/L biomass was measured in the upper level, while at a
depth of 10 cm, 150 mg/L biomass was still found. The
reduction
of
algae biomass observed
in
the covered cul-
tures was evidently due to settling of individual Sc. di
morphus
since
no
flocculation agent was observed under
the microscope. This type of settling was also described
by Conway and Trainor.13 The pH of the covered culture
increased only by
0.4
pH units to pH 7.4, evidently due to
C02 evolution in order to attain equilibrium under the
corresponding partial pressures. The results suggest that
the autoflocculation phenomenon is light-dependent, evi-
dently due to continuing intensive photosynthetic activity
after the autoflocculation initiation.
Effect of pH
Consistently, algae autoflocculation in outdoor auto-
trophic cultures was observed when culture pH was raised
due to photosynthesis. The direct effect of the pH
o n
algal
autoflocculation was confirmed by simulation of the pro-
cess through artificial pH changes in a standard jar test.
The pH of each
1
L outdoor autotrophic algal culture
was adjusted in the range 2.5-10.5 by addition of acid or
base prior to the jar test procedure. Figure 3 shows the
percentage of algae biomass removed from the outdoor
culture after flocculation and sedimentation at various
pH values.
A low removal peak was observed at pH 3.0. At that
point, algae are known to have zero net surface electric
chargel2-I4 nd the probability of contact interactions be-
tween cells increases since no or low mutual electric repul-
sion forces are present. No flocculation was obtained for
pH 5.0-7.5 while above pH 8.5, a flocculation zone was at-
tained and almost 98 of the algae biomass
was
removed
from the culture. This flocculation zone corresponds to
the autoflocculation observed in outdoor cultures. When
100
60
r
0
E
a
a 4 0
a
D
-
2 0
0
2 3 4 6 7 8
9
10
P H
Figure
3.
door culture after flocculation and sedimen tation
at
various
pH
values.
The percentage
of
Sc. dimorphus biomass removed from out-
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algal biomass was harvested from culture medium by cen-
trifugation, washed, and resuspended in distilled water
prior to the jar test procedure, this alkaline flocculation
zone was not observed. Resuspension in the original
growth medium or fresh modified medium restored the
flocculation ability in that alkaline zone. The results indi-
catethat he culture pH is an important factor in autofloc-
culation while algae photosynthetic activity is only a vector
to achieve the alkaline conditions. Furthermore, the im-
portance
of
the medium chemical composition for the
autoflocculation phenomenon is strongly suggested.
Autoflocculation Mechanism-Laboratory Stud y
Effect of Specific Ions
From the preceding experiments and from theoretical
considerations it becomes obvious that three chemical
ions ubiquitous in algae media are responsible for auto-
flocculation, namely magnesium, calcium and phosphate
ions. At stressed alkaline conditions, magnesium hydrox-
ide precipitates and constitutes the most active autofloc-
culation agent.
5
Since autoflocculation was observed to
take place under lesser alkaline conditions where magne-
sium hydroxide was absent , it was hypothesized that cal-
cium phosphate could be the active autoflocculation
agent and the experimental work herein was aimed in
confirmation of this phenomenon.
The effect of the above-mentioned ions
on
autofloccu-
lation was examined by standard jar tests which simu-
lated the natura l process by adding NaOH to achieve pre-
selected pH values. Figure 4 shows this effect, with the
addition of 2.0mM magnesium and alternatively 2.0mM
calcium ions while a concentration of
0.2mM
orthophos-
phate was present in the medium,
on
algal flocculation at
various pH levels. Calcium caused flocculation at pH
8.5
0 5
E
c 0 . 4
u
e
-
o 0.3
e
5: 0.2
u
c
4
0.1
-n
0 -
0
I
I
0
o
7.0
0.0
9.0 10.0
11 0
12.0
P H
Figure4.
pH manipulat ion. Ini tial or thophosphate concentrat ion was 0 2 m M
Effect of me dium com position on Ch.
vulguris
flocculation by
and above, while magnesium caused flocculation only
above pH 10.5. In the absence of phosphate, 2.0mM cal-
cium did not cause any flocculation over the entire alkaline
range. The same negative results were observed when a
concentration of 0.2mM was maintained but with the ab-
sence
of
calcium ions. The presence of phosphate ions had
no effect on flocculation with magnesium hydroxide at pH
levels above 10.5. The results indicate the importance of
calcium together with orthophosphate ions for effective
flocculation at the range of pH between
8.5
and
10.5.
Critical p H Value for Flocculation
Jar test sets which operated with algae suspension in
fresh Bolds medium containing an array of initial cal-
cium and orthophosphate concentrations demonstrated
an inverse relationship between the concentration of these
ions and the lowest pH value where flocculation was at-
tained, as shown in Figure 5. The pH value where 50 of
the algal biomass was removed from the suspension,
through flocculation and sedimentation, was defined as
the critical pH value for flocculation, (pHc), and it is in-
dicated schematically in Figure
6 .
The value of pHc for
flocculation in the corresponding calcium and orthophos-
phate initial concentrations shown in Figure
5
are given
in Table
111.
In the presence of 2.5mM calcium and
0.05mM
orthophosphate, the critical pH value for algal
flocculation is 9.3 and decreases to 8 . 3 when the ortho-
phosphate concentration increases to 0.21mM. The pHc
-
E 0.6
z 0 . 4
C
a2
O
n
0.2
a
VI
It
1 7 1 7
O r t h o p h o s p h a t e rn M
0
-
0 .05
0
-0.10
- 0 . 2 1
0 - 0 . 4 2
7 0 8 .O 9 0
10.0 11.0
P H
Figure
5. Effect of di f ferent or thoph osph ate concentrat ions at 2 . 5 m M
calcium on s imulated algal autof locculat ion. A rrows indicate pHc val-
ues
for the dif ferent exper imental c ondi t ions .
Figure
6.
Percentage of algae removal by pH m anipula t ion dur ing a
s imulated autof locculat ion tes t and the def ini t ion
of
the cr i t ical pH
value for the flocculation.
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Table
IU. The flocculation critical pH values pHc) for
Ch.
vulgaris culture
at initial 2.5mM calcium and various orthoph osphate concentrations.
Ort h-P concentration Flocculation critical pH
d)
PHc)
0.05
0.10
0.21
0.42
0.95
9.3
a 7
8.3
8 .0
7.2
value was found to be a useful and a reliable parameter to
quantitatively define the flocculation process.
The combined effect of initial concentrations of cal-
cium and orthophosphate on the flocculation critical pH
value of an algae suspension is shown in Figure 7. As the
calcium and orthophosphate concentrations increased,
the pHc values decreased and, at high concentrations, al-
gae could be flocculated almost at neutral conditions. For
concentrations of 0.1mM-0.2mM orthophosphate and
1
S mM- 2 .5mM
calcium the flocculation critical pH
value were between 8.4 and
9.0.
These alkaline condi-
tions can be easily attained by photosynthetic activity of
an algal culture when carbon is not sufficiently available
as was the case in the outdoor autoflocculation experi-
ments described earlier in this article.
The Chemical Precipitant
Results of a laboratory jar test showing changes in al-
gae concentration in suspension as well as algal zeta po-
tential, are given in Figure 8(a), while the concentrations
of dissolved calcium and orthophosphate ions remained
after flocculation and sedimentation are given in Figure
8(b). As seen from Figure 8(a), algal flocculation was at-
tained above pH 8.5. The algae surface electric charge as
indicated by Z p measurements was apparently neutral-
ized toward pH 9.0 while above that pH value a positive
surface charge was measured. The simultaneous removal
Figure
7. Th e combined effect
of
initial calcium and orthophosphate
concentrations on flocculation critical pH value
pH c)
of
Ch.
vulguris
culture.
c = I 1
6
200
P
I
I 5
c
N
c
-15
N
7 0 8 . 0 9.0 10 0 11.0
PH
2.0 p
1.5 a
W
0
.-
1
r.0 a.0 9.0 10 0 11.0
P H
Figure 8. Simulated algal autoflocculation: a) algae concentration
and cells zeta potential, and b) dissolved calcium and orthophosp hate,
after flocculation and sedim entation.
of calcium and orthophosphate ions [Fig. 8(b)] indicates
that under alkaline conditions a precipitate of calcium
phosphate is formed. The formation of such chemical pre-
cipitate which constituted an agglomerating agent be-
tween the algal cells was observed using light and electron
microscope and was reported in previous work.16 Fur-
thermore, the chemical composition of the precipitate
was studied in details using an electron microscope x-ray
dispersive analysis. It showed conclusively that calcium
and phosphorus were indeed the main elements in the
precipitate. An electrophoretic analysis of algal free cal-
cium phosphate precipitate showed that this precipitate
has a positive surface electric charge. Evidently, this posi-
tively charged precipitate may be adsorbed to and react
with algae cells to neutralize their negative surface elec-
tric charge and thus promote algal flocculation.
CONCLUSIONS
Autoflocculation of microalgae is attained in outdoor
autotrophic cultures when carbon dioxide limitation
is
in-
duced. The process is associated with algae intensive
photosynthetic
C 0 2
consumption from the carbonate
system that causes the pH to rise. Experimentally, auto-
flocculation can be simulated by chemically induced alka-
line conditions.
A
quantitative reliable parameter, flocculation criti-
cal pH (pHc), was defined and used to assess the floccu-
lation performance of algal cultures and to identify the
relevant chemical ions of autoflocculation in the medium
and their concentration.
The presence of calcium and orthophosphate ions in
sufficient concentrations prior to the autoflocculation ini-
tiation was found to be crucial for the process. To attain
autoflocculation within the pH range 8.5-9.0, the culture
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should contain 0.1mM-0.2mM orthophosphate and be-
tween
1
OmM-2.5mM calcium.
A
reduction in calcium
and orthophosphate ions from the solution is attained as
these ions become stoichiometrically part of the floccu-
lated matter.
Based on the experimental results and on theoretical
considerations, the following autoflocculation mechanism
is postulated. Increasing pH in algal culture either by
COZ
consumption by algal photosynthesis or by direct addition
of alkaline leads the culture medium to a supersaturation
state with respect to calcium and phosphate ions. Such
supersaturation causes an initial nucleation of calcium
phosphate precipitation which is promoted by the algal
cells serving as solid surface. In the presence of excess cal-
cium ions, the calcium phosphate precipitate is positively
charged and therefore adsorbed and reacts with the nega-
tively charged algal cells to agglomerate them and pro-
mote algal flocculation.
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AND SHELEF: ALGAL AUTOFLOCCULATION
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