Archives of
Arch Microbiol (1984) 139:421-426
Phormidium J-1 bioflocculant: production and activity*
Ali Fattom and Moshe Shilo
Division of Microbial and Molecular Ecology, Life Sciences Institute, The Hebrew University of Jerusalem, Jerusalem, Israel
Abstract. Phormidium J-l, a benthic filamentous cyanobac-
terium, isolated from a drainage channel, was found to
produce a high molecular weight polymer. This substance
can flocculate bentonite particles from suspensions. At the
stationary phase of growth the cells excreted this
bioflocculant into the surrounding medium. Production was
enhanced by reduction of the calcium content in the growth
medium or by increasing its EDTA content. Above the
isoelectric point (pH 3.5) the bioflocculant is negatively
charged. The presence of minimal concentrations of divalent
cations in the reaction mixture is required for its flocculating
activity. The production of bioflocculant could be of great
importance in clarification of turbid water bodies, thus
allowing light penetration to the sediment/water interface.
Bioflocculant production and excretion was not found in
several other benthic cyanobacteria.
Key words: Bioflocculant -- Benthic cyanobacteria
Introduction
A major limiting factor in the growth of benthic cyanobac-
teria, by far, is the amount of light reaching the soil-water
interface. Light limitation is imposed, first, by the absorp-
tion of radiation in the water column overlying the bottom.
This light absorption is even more marked in turbid waters,
rich in suspended particles and plankton. In stratified lakes,
layers or plates of such microorganisms may create, in effect,
light filters. Furthermore, the continuous sedimentation of
particles and agglutinated cells from the water column cover-
ing the benthos imposes a dynamic stage of changing light
conditions. It has been found that often only 1% of surface
light intensity penetrated to 2 - 4 mm in the sediment
(Fenchel and Staarup 1971).
Under such conditions, mechanisms to secure minimal
light requirements for survival and growth are crucial for
the benthic cyanobacteria. Since such cyanobacteria formed
the most ancient fossils found already in the Precambrian
stromatolites (Schopf 1974), they must have adapted to the
benthic habitat with its light limitations early in their evolu-
tion.
* Dedicated to Prof. Dr. H.-G. Schlegel on the occasion of his 60th
birthday
Offprint requests to. A. Fattom
Micrebiology
9 Springer-Verlag1984
Several adaptive and metabolic strategies have been
shown in these organisms for overcoming the light
limitations. One is a positive phototactic gliding motility
(Castenholtz 1982). In addition, chromatic adaptation and
increased synthesis of photosynthetic pigments (Bogorad
1975) have been found.
Another possibility for increasing the amount of ambient
illumination at the benthic surface interface is to reduce the
turbidity of the surrounding waters through the excretion
and action of a flocculating substance. Precipitation of clay
particles and clarification of a major drainage channel in the
Huleh swamp caused by production of a sohible flocculant
has been described (Avnimelech et al. 1982; Zur 1979).
This paper describes the production and activity of a
bioflocculant from Phormidium sp. strain J-l, a benthic
cyanobacterium, isolated from the Huleh drainage channel.
Preparation of the excreted and cell-bound bioflocculant
extracts is described in this paper, as well as their quantifica-
tion and the conditions required for bioflocculant activity.
Materials and methods
Cyanobacterial strains and culture conditions
Phormidium sp. strain J-1 was isolated from a drainage
channel in the Huleh swamp in Israel and obtained in axenic
culture. The strain was deposited in the American Type
Culture Collection and given the number ATCC 39161.
Other cyanobacteria together with their growth conditions
and culture media are as described by Fattom and Shilo
(1984).
Preparation of cell-bound and extracellular bioflocculant
Bioflocculant preparations were prepared from cyanobac-
terial cultures grown under standard conditions (Fattom
and Shilo 1984) or as noted in Results. The preparation
process is shown in the flow chart (Fig. 1) and included
pronase treatment and ethanol precipitation. Extracellular
bioflocculant secreted into the growth medium was concen-
trated in Fraction A. Fraction B represents cell-bound
bioflocculant, which was obtained after disintegration of
washed cells suspended in 10 ml, 20 mM Tris buffer, pH
7.0, supplemented with 10 mM MgSO4, in a Braun cell
disintegrator (Melsungen Model 53030) for two minutes
with 5 g of Ballotini glass beads (0.1 mm C5). Both fractions
were lyophilized and stored at room temperature.
422

Culture
centrifugation

Cell pellet Supernatant

~Tris|resuspendedbuffer
in vacuum
levaporation
Cell suspension Concentrated supernatant 0.5-
lcell disintegration (10% in initial volume}
|pronase, treatment,
Cell homogenate 88
Dialysate
ethanol pre- I I I A/ X
Cell debris Filtrate Jc[pitation 0 25 50 I O(
|pronase treat- ~ o n Bioflocculant Concentration (~g dry weight/ml)
discard ~ment, dialysis
Dialysate Pellet Supernatant Fig. 2. Assay of extracellular (fraction A) Phormidiumbioflocculant.
ethanol
cipitation,pre- Fraction A Fraction C
Bentonite was added to give a final concentration of 600p.g per ml
Bioflocculant
assay mixture. T is the time at which 50% of initial optical density
~ ugat on was observed
Pellet Supernatant
FracEan B Fraction D
biofiocculant
Fig. 1. Flow Sheet for separation of the bioflocculant of 0.5 )l,
/ \
Phormidium. Culture and ethanol precipitates were centrifuged at I x
\
10,000 x g for 10 min. Vacuum evaporation was done at 45~ pro- I ,,
nase treatments with 100 gg pronase (Sigma) per ml at 37~C for 1 h. g -
Dialysis was done overnight at 4~ against distilled water, ethanol
9 \\0~
precipitation at 0~ with added pure ethanol to give 66% final
/ \
-\ 0.25 I IL
concentration. Cell pellets were resuspended in 0.02 M Tris buffer
/ \
(pH 7) supplemented with t0 mM MgSOr and cell disintegration [ \~,
was carried out with 5 g glass beads (0.1 mm diameter) per 10 ml / '-.
suspension, chilled in ice and agitated 2 min in a Braun cell
distintegrator (model 53030, Melsungen). Cell homogenate filtered
I I I
through GF/C Whatman filter
0 1.2 2.4 :5.6 4.8
Benlonite (rag / ml)
Fig. 3. Bioflocculant activity as function of bentonite concentration.
Assay of bioflocculant activity Standard bentonite to different final concentrations was added to
the assay mixture containing 25 gg fraction A per ml. T is time at
The activity o f the various bioflocculant preparations was which 50% of initial optical density was observed
determined by the rate of settling of standard bentonite
particles (Fisher) in an aqueous suspension. Bentonite was
added to Klett tubes to give bentonite concentration of
600 mg/ml. The tubes contained samples of extract dissolved The time required for precipitation of the bentonite
in 2 m M Tris buffer (pH 7.5) supplemented with 1 m M depended on the concentration of the bentonite as well
MgSOr to give a final volume o f 5 ml of assay mixture, the (Fig. 3). A n excess of either bioflocculant (Fig. 2) or ben-
tubes were shaken and allowed to stand at r o o m temperature tonite (Fig. 3) depressed and, at the higher concentrations,
(26 ~C). Turbidity o f the assay mixture was measured in a even prevented flocculation within the test period.
Klett-Summerson calorimeter (filter 54). The time required At lower bioflocculant concentrations (up to 12 gg per
for a 50% decrease in initial turbidity was taken as the ml), the flocculating activity was linear with respect to time
measure of flocculant activity. Spontaneous decrease to 50% required for the 50% reduction in initial turbidity (Fig. 2).
of initial turbidity of the bentonite in the test buffer without The unit of bioflocculant activity was therefore defined as
bioflocculant took more than 30 min at this temperature. that flocculant concentration per ml test solution (in the
linear range) that produced a 50% decrease in initial ben-
tonite turbidity within 5 min at the standard bentonite con-
Chemical determinations centration of 0.6 mg per ml test solution.
Proteins were determined according to Lowry et al. 1951. Bioflocculant activity was not affected within the p H
range of 4 to 10. Below pH 4 and above p H 10, there was
spontaneous and rapid flocculation of the betonite particles,
Results so that the effect could not be tested.
Temperature affected the bioflocculant activity. Fig. 4
Bioflocculant activity of Phormidium sp. strain J-1 shows that activity markedly increased with a temperature
The activity of the extracellular bioflocculant (Fraction A) rise to 55 ~C. At 80 ~C (not shown in the figure) the floccula-
was tested in the bentonite assay system. When the bentonite tion proceeded even more rapidly.
concentration was kept constant (Fig. 2), a sharp peak of The presence of cations was found to be essential for
maximal activity was observed at a given bioflocculant con- bioflocculant activity. Magnesium and calcium cations were
centration (in this case, 25 ~tg Fraction A per ml test twelve times more efficient than the monovalent sodium ion.
mixture). Figure 5 shows that while 0.8 m M Mg 2+ sufficed to achieve
 423

50C At B 5OO
/
2( .... e-----/--e
jibe
E
E /
ff ,/ / /
/ / 100 . ~
,/ /
11 ,/ //
.~ 30C i /
/ /
~d
/ I0 / 1/
8 / i
iI 8 i/~.* . . . . . . . & /
LL iI d" !

._~ /I r;
/" / ,4
/ I
Lj ,- . . . . . . . 9

/ / fl~ / /
/ i/ rn /. /
Y ~oo
o~ ~ i i
5 IO 15 5 IO 115
Age of Culture(d)
115 i i ; i Fig. 6 A, B. Production of ce]l-bound and extrace]lular bioflocculant
25 35 4 55 T~
T~ as a function of the age of the culture in complete modified BG11
Fig. 4. Effect of temperature on extracellular bioflocculant activity. medium and with reduced cation concentrations or with addition
Assay mixtures contained 0.3 bioflocculant units of fraction A per of EDTA. Phormidium J-I was innoculated in modified BG11 (part
ml. Results expressed in percent of bioflocculant activity obtained B of the graph) ( 9 into BG11 with reduced calcium (1/100 of BGll
at 26 ~C content) (part A of the graph) ( ~ ) and into BGtl medium with
increased EDTA (100 x that of the original medium) (part A of the
graph) (A). Cells were incubated under standard growth conditions
and at different intervals the cell protein (@ 4> A), extracellular
Ioo
bioflocculant ( 9 O A), and cell-bound bioflocculant (@ @ A )
were measured

EJ
100 ,_!
g
o 80 o
9

,-c ~.~- 75
~._>

~c 6o ~S_ 50
c0
9"6
<50 g~
0.2 0.6 I0 2 [Mr
89 4 6. . 8 . .i0 12 '\ 20[Na*]
Ct]fion Concentration (mM)
'
-o
c

Fig. 5. Effect of cation concentration on extracellular bioflocculant
activity. The standard assay mixtures containing 5 units of fraction
A per ml were tested with different concentrations of MgSO4 (@)
and NaCI (0). Results are shown in percent of standard assay
(containing 0.6 mM MgSO4 and 6 mM NaC1) activity
m a x i m u m flocculant activity, 12 m M N a + was required for
similar effect.
Extracellular and cell-bound bioflocculant were pre-
pared and tested from Phormidium strain J-I cultures in
different growth media and at different stages o f growth.
During the exponential growth phase in the modified B G
11 medium, cell-bound bioflocculant (Fig. 6) steadily in-
creased, but there was no demonstrable accumulation o f
extracellular bioflocculant. Bioflocculant a p p e a r e d in the
extracellular fraction o f the culture only after the cells
reached the stationary phase. Figure 7 shows that
bioflocculant accumulated in the cells during exponential
growth was excreted and accumulated in the growth m e d i u m
as the cells age.
W h e n the calcium content o f the modified B G 11 me-
dium was reduced to a h u n d r e d t h o f its usual concentration,
stationary-phase extracellular bioflocculant increased by
50% and cell-bound flocculant by 30% (Fig. 6A) c o m p a r e d
o
A21
25
/ 9
o 0 t I
4 8 12 16
Time (d)
Fig. 7. Distribution of bioflocculation activity in Phormidium J-1
cultures. Extracellular (open symbols) and cell-bound (block
symbols) bioflocculant was measured (see Materials and methods)
in three growth conditions EDTA x 100 (A), Ca/100 (O) and modi-
fied BGtl (O)
to the results shown in Fig. 6 B. A similar effect was obtained
by increasing the E D T A concentration o f the growth me-
dium a hundred-fold (Fig. 6 A). This increase in extracellular
bioflocculant was not due to a leakage or lysis o f the cells,
since no cellular components, such as chlorophyll a or
phycocyanin, were seen released into the medium.
Reducing nitrate to 2% and p h o s p h a t e and sulfate to
i % o f the original concentrations in the complete modified
BG 11 medium did not increase biofloccutant p r o d u c t i o n
and even caused some reduction (Table ~).
Bioflocculant production in other cyanobacteria
The p r o d u c t i o n o f extracellular a n d o f cell-bound biofloccu-
lant in other cyanobacterial strains from various
424

Table 1. Production of extracellular and cell-bound bioflocculant Table 2. Formation of bioflocculant by different cyanobacterial
by Phormidium sp. strain J-1 under conditions of mineral starvation species

Bioflocculant units a per ml culture Bioflocculant units b per ml culture

Extracellular Cell-bound Extracellular Cell-bound Cellularprotein

(fraction A) (fraction B) (fraction A) (fraction B) (mg/ml-1)

Complete BG11 9 5 Cyanobacterial

Low in nitrate species
(2% ofComp BGtt) 6 4 Phormidium sp.
Low in phosphate strain J-1 a 50 16 400
(1% of standard BGll) 4 1.5 Anabaenopsis
Low in sulfate circularis a
(1% of standard BGll) 3 4.5 strain 6720 1 ~ 1 200
Oscillatoria
" Extracts prepared from Phormidium J-1 cultures grown in respec- limnetiea a 1 ~ 1 500
tive media under standard conditions for 14 days were extracted
as in Fig. 1 and were tested as in test assay for bioflocculation Plectonema
unit described in Results. Cellular protein at the harvest time was boryanum ~
400 ~tg per ml strain 6306 1 2 520
P. boryanum
strain 594c - ~ 0.6 300
Spirulina
environments was tested. Table 2 indicates that biofloccu- tenuis ~ 1 ~ 1 500
lant production was not common to all the cyanobacteria Anaeystis
and that varying amounts were produced by different nidulans
strains. Bioflocculant production was highest in the strain 6311 c - ---<1 550
P h o r m i d i u m sp. strain J-l, which was found in nature to be
a Cultures were harvested after 28 days' growth and bioflocculant
responsible for the clarification of the water in the Huleh
fractions prepared as in Fig. 1
drainage channel (Avnimelech et al. 1982; Zur 1979). b Bioflocculant unit as described in Results
c Cultures were harvested after 20 days growth
The cyanobacterial strains were grown in the appropriate medium
and under the same conditions for four weeks. The cells were
Discussion harvested by centrifugation at t0,000 x g for 10 min, and cell-bound
and extracellular bioflocculation activities were recorded as
Studies of the water drainage channels of the Huleh Valley mentioned in Materials and methods
showed dramatic difference in the water clarity of one of the
channels. It was found that the water in the western channel
contained fewer amounts of suspended particles compared
with the high content in the water of the eastern channel. In the case of benthic photoautotrophic cyanobacteria
These observations suggested that some sort of clay aggrega- the flocculant action in shallow water bodies might have a
tion leads to sedimentation of the suspended load. Zur critical effect in allowing light to reach the sediment/water
(1979) found that a benthic filamentous cyanobacteria was interface. Another yet untested effect might be the flocculant
dominant in the clear drainage channel, forming a continu- enhanced sedimentation of particles carrying absorbed
ous lawn at the channel bottom and suggested that an nutrients and their accumulation at the water/soil interface
extracellular substance produced by the cyanobacteria, where they would be more readily available to the benthic
could be responsible for the flocculation activity toward the organisms. Thus, in oligotrophic conditions, the benthic
suspended clay. niche would be continuously enriched.
We isolated and purified the dominant cyanobacteria The P h o r m i d i u m bioflocculant is cell-bound during the
from the western channel. It was designated as P h o r m i d i u m exponential phase of growth, but a large portion of
J-1. The axenic cultures of Phormidium J-1 were found to bioflocculant is released at the stationary phase. The en-
produce a polymeric substance which flocculates suspended vironmental conditions may be involved in the control of
bentonite particles. its production or release. Production of bioflocculant was
Aggregation and flocculation of suspended particles by increased by calcium starvation or by increasing the EDTA
microbes are of great ecological significance, and have been concentration in the growth medium. Out of a whole series of
extensively discussed in the literature (Harris and Mitchell nutrients, including nitrate, phosphate, sulfates, and certain
1973). These phenomena play an important role in many other cations, only reduction of calcium affected the
systems such as open lakes, seas and waste treatment. Pro- bioflocculant production, leading to increased levels of both
duction of cell-bound and extracellular polymers may pro- cell-bound and extracellular flocculant. This effect may have
tect cells from the toxic effect of metal ions in the environ- some importance in the natural environment.
ment (Dugan 1975). On the other hand, extracellular It seems that the P h o r m i d i u m bioflocculant is a
polymers can play a role of chelating agents, enriching the macromolecule, since it was retained in the cellophane bag
microorganisms with minerals such as iron (Trick et al. even after prolonged dialysis. Preliminary chemical analysis
1983). of the polymer showed that it contained polysaccharides,

IOO _ ~- --~ - -//- ~-
/ bentonite suspensions flocculate only above critical concen-
/
._>
/ trations of MgC12 at these concentrations.
~'6 50 _tI
I
I
I
,J. n I //
0.5 I 3
Pore Size (.,u m)
Fig.8. Filtrability of bioflocculant. Freeze-dried bioflocculant from
Phorrnidium J-1 cultures was dissolved in Tris buffer (0.002 M)
supplemented with 1 mM MgSO~ at a concentration of 5 units/ml
and filtered through millipore filters with different mean pore size
fatty acids and protein moieties. Above the isoelectric point
(pH 3.5) the bioflocculant is negatively charged. Gel filtra-
tion (ACA22, LKB) of the polymer showed that it had
a molecular weight > 2 x 106. Filtration through Millipore
membrane filters indicated that the polymer produced
micelles of > 0.2 gm in diameter (Fig. 8).
Phormidium bioflocculant activity showed optimum
activity depending on both the bioflocculant and bentonite
concentrations. No flocculation occurs when an excess of
either substance is present. This phenomenon, with respect
to excess flocculant, so-called steric stabilization, has been
proposed by a number of authors, Lyklema 1968; Osmond
et al. 1975; Theng 1979; Van Olphen 1963 and Vincent 1974.
We found that between pH 4 and 11 bioflocculation is
not affected. These results correlate well with the chemical
properties. Above the isoelectric point (pH 3.5), the
functional groups were ionized and the bioflocculant nega-
tively charged. On the other hand, activity is affected by
changes in temperature. Increased temperature causes an
increase of entropy and breakage of the hydrogen bonds
inside the molecule, resulting in an exposure of more binding
sites of the polymer.
The degree of flocculation at any particular concentra-
tion of polymer depends on a) the length and number of
extended segments and b) the available surface onto which
extended segments can bridge (Michaels 1954).
Two main theories have been suggested to explain the
flocculation of clays by polymers from suspension. One
theory is the flocculation by a bridging mechanism. If the
polymer is long enough and has a sufficient number of free
sites which can bind to more than one particle, it will act as
a bridge (Michaels 1954; Fleer and Lyklema 1974). The
other theory suggests that flocculation is due to a change in
the charge density (Sarker and Teat 1973). Experiments with
polymers having the same charge as those colloid particles
have been conducted. It was found that a certain min-
imum concentration of cations in solutions is necessary
before flocculation can take place. Accumulation of
cations reduces the effective charge density on the surface
of the particles and allows a closer inter-particle approach
until attraction forces overtake (Michaels 1954; Parfitt and
Greenland 1970).
In the case of Phormidium biflocculant it is difficult to
determine which of the two theories better explains its activ-
425
ity. Assuming that cations form complex bridges between
negatively-charged particles (Santoro and Stotsky 1967) the
presence of cations in the polymer-clay system is essential
--o if flocculation is to occur. We found that bioflocculant-
Experiments with a number of cyanobacteria showed
that Phormidium sp. strain J-I is the only species tested so
far which produced large amounts of bioflocculant. These
amounts appear to be sufficient for clarification of the water
column in natural turbid-water habitats (Avnimelech 1982;
I I Zur 1979). In view of the potent bioflocculant activity of
5 Phormidium it will be of interest to determine to what extent
this capacity could be exploited in clarification of water
bodies or in production of an industrially important
bioflocculant.
Acknowledgements. This work was supported in part by the Wolfson
Foundation and by Solmat Systems Ltd. We thank Dr. Y. Elkana
for her help in the gel filtration experiments.
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Received March 9, 1984/Accepted April 19, 1984