Algal AutofIocculation Verification

7/26/2019 Algal AutofIocculation Verification

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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

John Wiley Sons, Inc.

CCC

006-359218410201 2-06 04.00


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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.

S U K E N I K A N D S H E L E F : A L G A L A U T O F L O C C U L A T I O N

143


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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-

144

BIOTECHNOLOGY AND BIOENGINEERING VOL. 26, FEBRUARY 1984


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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.

SUKENIK AND SHELEF: ALGAL AUTOFLOCCULATION

145


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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

146

BIOTECHNOLOGY AND BIOENGINEERING VOL. 26, FEBRUARY

1984


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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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J. Oswell, CRC Handbook of Microbiology

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SUKENIK

AND SHELEF: ALGAL AUTOFLOCCULATION

147

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Algal AutofIocculation Verification