Note: Descriptions are shown in the official language in which they were submitted.
FIE~D OF THE INVEN_IO
This invention relates to the production and use of
algae as a source of polymeric materials displaying strong
flocculating activity. An important feature of the invention
involves the discovery that the growth of algae can be regulated
so as to favor the production of large amounts of flocculants,
useful in waste water treatment operations for the breakdown and
removal of solids, reduction of BOD, and the breakdown of grease
blankets. The flocculants are also useful as soil conditioners
improving soil tilth, improving aeration, drainage, moisture
retention, root development and have utility for other applica-
tions where flocculating agents are commonly employed, such as in
drilling fluid technology as drilling mud extenders. As is known
in the art, products exhib~iting flocculant activity in higher
.....
';~`' ~, . '
:
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concentrations are also useEul as surfactants, detergents,
emulsi~iers and dispersants and products formed in accordance
with the invention are of utility for such purposes in appro-
priate concentrations.
BACKGROUND OF THE INVENT~ON AND PRIOR ART
The production of algae, as a protein rich food source
for animals and humans, as well as a source of other valuable
products such as dyes, vitamins and the like is extensively
reported in the Carnegie Institution of Washington Publication
No. 600, ALGA~ CULTURE FROM LAB~RATORY TO PILOT PLANT, Edited
by John S. Burlew and published at Washington, D.C. in 1964.
This publication contains studies of the various factors in-
volved in obtaining high yields from cultures of algae, focusing
primarily on the species Chlorella pyrenoidosa but of applica-
bility to other species of algae as well. In addition, the
above publication and other prior art including U.S. Patent
No. 2,732,661 disclose the cultivation of algae under conditions
which cause a predominance of intracellular protein, lipid
o~ carbohydrate by regulating the amount of available nitrogen.
Production of algae as a source of proteins ana lipids, and
other materials derived ~rom the algae, are discussed in the
Carnegie publication.
; According to another body of prior art, utilization of
bacterial polysaccharides as flocculating agents, especially
for aggregating soil particles, thereby improving 50il structure
is known. U.S. Patents such as No. 2,780,888 and No. 2,901,864
teach the application of these bacterially produced biopolymers
to the soil as a means for promoting soil aggregation, thereby
producing a granular structure which is sufficiently porous
A~ ~,?
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to allow air, water, and plant roots to penetrate through
the soil. According to these patents, sucrose as a raw material
is converted to dextran by innoculating a nutrient medium
containing sucrose with a dextran synthesizing bacteria such
as Leuconostoc mesenteroides. The dextran may be used in
granular form or in solution in an aqueous medium and applied
to soil.
In addition to the foregoing, long chain synthetic polymers
useful as soil conditioning agents which are capable of aggre-
gating soils and useful for other applications where flocculatingactivity is required, are disclosed in the art. Examples
of synthetic polymeric materials useful for increasing aggre-
gation in surface soil are disclosed in Hedrick et al U.S.
Patent No. 2,651,885. According to the Hedrick et al patent,
water soluble polymeric electrolytes having a molecular weight
of at least 10,000, including polymers of acrylic acid, co-
-` polymers of maleic anhydride and the like are provided. These
polymeric materials are effective in improving soil structure
but their use has been somewhat limited in view of their high
cost.
SUMMARY AND OBJECTS OF THE INVENTION
With the foregoing in view, it is an object of the present
invention to provide a new and improved method for producing
flocculating agents by the cultivation of algae.
Another important object of the invention is the pro-
vision of an economical method of producing flocculating agents,
4~1Z2
requiring as raw material algae, llght, a source of assimilatable carbon and
common plant nutrients.
Basically considered, the inventlon involves the discovery that al-
gae can be made to favor *he production of flocculating agents by limiting the
cellular nitrogen. More specifically, it has been discovered that when an
algal culture has used up its available nitrogen supply so that the cells are
deficient in nitrogen, and when other nutrients in the medium are available
in sufficient quantity that they are not limiting growth factors, the cells
will favor the production of high molecular weight extracellular polymers ex-
hibiting strong flocculating activity.
According to one aspect of the invention, there is provided a methodof producing biopolymers exhibiting flocculating activity comprising; cultivat-
ing algae in the presence of light and carbon dioxide in a nutrient medium suit-
able for exponential growth until a desired population density is reached,
thereafter restricting the available nitrogen and continuing to cultivate the
sufficient algae while maintaining a supply of available phosphorus and other
plant nutrients in the culture medium as required so that the nitrogen content
of the cells drops to below about 5% by weight, and after the cellular nitrogen
is below 5~ by weight, withdrawing active flocculating agent from the culture
medium.
Another aspect of the invention provides a method of producing algal
biopolymers having flocculating activity comprising, cultivating an algae in an
aqueous nitrogen rich nutrient medium in the presence of light and carbon diox-
ide until the culture reaches the deslred density, thereafter continuing the
cultivation until the cellular nitrogen is below about 5%, and thereafter with-
drawing the flocculant active polymers.
A further aspect of the invention provides a method of producing
polymeric material having flocculating activity from algaeJ comprising the steps
of culturing algae in a nutrient medium under conditions promoting optimum growth
for a first period of time until a predetermined cell density is reached, there-
after culturing the algae for a second period of time~ depriving the algae of
available nitrogen during the second period of time to reduce the cellular nitro-
~ -4-
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z
gen content to below about 5% by weight and maintaining light, C02 and plant
nutrients other than nitrogen in quantities which are not growth limiting dur-
ing the second period of time.
A further aspect of the invention provides a method of producing a
flocculating agent which comprises culturing algae in a tank containing an
aqueous nutrient medium having sufficient available nitrogen, other nutrients,
and C02 in the presence of light, so as to foster exponential growth of the
algae, when a predetermined cell density is reached, transferring a portion
of the algae and nutrient medium to a second tank, adding water to the second
tank in amount proportional to the volume of algae and nutrient medium trans-
ferred and thereby reduce the percentage content of nitrogen in the medium, and
continuing the culturing in the second tank until a predetermined cell density
is reached.
Although the algal produced polymers have not been fully identified,
these polymers are ethanol precipitable, Anthrone sugar reacting materials and
hence are apparently polysaccharides. Filtration through caIibrated membranes
indicates
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that these agents having a molecular weight of approximately
100,000 daltons. In tests, flocculant activity is not destroyed
by proteolytic enzymes indicating that the polymers are not
protein in nature. The flocculants produced according to the
invention are primarily extracellular metabolites although with
certain species, flocculating activity is to some degree also
associated with the substance of the algal cells. From a
functional point of view, the flocculating agents produced by
the invention resemble the bacterial polysaccharides and
synthetic polymeric flocculating agents mentioned above.
Conditlons Eor optimizing yields of algae are now well
known in the art a~ rcported Eor examplel ln ~LGAL CU~'rUR~ FRO~
LAaORATO~Y TO P~LOT Pl.AN~, r0Eerred to abovo. ~a~icall~
considered, the algae requLre a sufEiclent carbon source usually
in the form of carbon dioxide, light as an energy source, a
source oE nutrients and favorable temperature conditions. The
nutrient medium does not dif~er materially from that used by
higher plants, consisting of an aqueous solution of fixed
nitrogen, other mineral nutrients and micro-nutrients. The
algae are grown in natural sunlight or artificial light which
may be supplied for example, by means of lamps sold by the
Sylvania Corporation under the Trademark Gro-lux.
When algae are cultured in a suitable nutrient medium with
the adequate light at favorable temperatures as explained in the
above-identified pulication, there will be an exponential phase
of growth in which there is a geometric increase in the number of
cells. As the culture continues to grow it reaches a point where
the rate of progres~ion ~lacks off and it thereaEter enters a
Z2
stationary phase in which theee is little or no increase ~n popu-
lation density, due mainly to the depletion of one or more of the
nutrients in the nutrient solution, the inability of light to
penetrate the culture, or the absence of an adequate supply of
carbon dioxide. In the production of flocculants, the invention
first involves the culturing of algae under conditions favoring
healthy growth so as to maximize yield. When a predetermined
cell density is reached as measured by the number of cells or
weight of cellular matter per unit volume of medium, culturing
is continued under conditions to be described herein so as to
favor production of flocculants. According to one form of the
invention, the culture is harvested from the initial or nurse
tank when it approaches its maximum density, that is, at or near
the end of the logarithmic stage of growth. Typically,
densities of from 1 x 107 to about 1 x 108 cells per ml, which
correspond to between about .200 to 2 grams of cellular matter
per liter are achieved before exponential growth ceases and
these densities are suitable from the standpoint of the pro-
duction of flocculants on a commercial scale. The culture is
then transferred to a tank where culturing is continued under
conditions favoring flocculant production. During this phase of
growth, culturing is carried out under conditions of nitrogen
deficiency. During exponentîal growth, when other growth
factors are non-limiting, cellular nitrogen is about 10% dry
weight. In contrast, flocculant production in large quantities
is observed when cellular nitrogen is below about 5% dry weight.
The invention is particularly applicable to the cul-
~ Q~ 2
turing of green and non-nitrogen fixing blue-green algae. Ex-
cellent results are obtained using certain green, unicellular
algae which are normal inhabitants of soil and fresh water. A
preferred genus is Chlamydomonas. Within this genus, cultures
of the species Chlamydomonas mexi_ana have been found to yield
exceptional results. A further example of an alga to which the
techniques of the invention apply is Chlorella, of which
Chlorella pyrenoidosa is exemplary.
-
When cultured according to the invention, Chlamydomonas
lo mexicana has been found to produce an active flocculating agentconstituting 80% of the total culture dry weight. The agent is
is stable under normal environmental conditions; no loss in
flocculant activity has been detected in cultures stored at room
temperatures for up to six weeks.
In carrying out the invention it is important that the
cultures be exposed to adequate light and be provided with an
ample supply of carbon dioxide during the flocculant producing
phase as well as the growth phase. During the flocculant pro-
ducing phase, it is preferred that the cultures be exposed to
light substantially continuously. Under conditions o-f continuous
light exposure, larger yields of flocculating agent are produced.
It is theorized that this is because the algae draw on their
carbohydrate reserve when light is not available to them thus
consuming or restricting the production of flocculating agent.
In the description which follows, reference is made
to the accompanying drawings in which:
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Figure 1 is a plot o culture growth, carbohydrate viscosity and
flocculation value as a function of time;
Figure 2 illustrates the relationship between ~locculation value and
cellular nitrogen;
Figure 3 is a plot of carbohydrate/cell and cell production versus
time;
Figure 4 shows plots of culture growth as measured by optical density
and viscosity for cultures grown on medium of different nitrogen concentrations;
Figure 5a illustrates one part of the apparatus shown in Figure Sb,
which illustrates a culturing apparatus;
Figure 6 is a flow chart illustrating a process for the production
of flocculants according to the invention;
Figure 7 illustrates flocculant values o cultures of Chlorella of
different cellular phosphorous contents;
Figure 8 illustrates flocculation value of cultures formed according
to the invention as compared with a polystyrene sulfonate flocculating agent;
and
Figure 9 illustrates the effect of applications of flocculants pro-
duced according to the invention to soil.
The examples which follow will serve as illustrative of the various
aspecks o the invention using the green alga species Chlamydomonas mexicana
and Chlorella py~enoldosa. In certain examples set out below, the nutrient
_
medium of Table I was employed for growth and flocculant production phases of
culturing.
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~ 4~ 2
TABLE I
KN03 .180 g/L
MgS04.7H20 .05 g~
C C 2.2H20 .166 g/L
KH2P04 .050 g/L
Trace Elements .12 ml/L
Iron EDTA .12 ml/L
Trace Elements Iron EDTA
H3B03 2.86 g/L Disodium EDTA 26.1 g/L
MnS4 H21.23 g/L FeS04.7H2024.9 g/L
ZnS4 7H2.22 g/~ NaOH (IN~263 ml/L
MoO3(85%).017 g/L Water to 1 L
CuSO~.5H20.079 g/L
CoCL2.6~20.041 g/L
Various nitrogen compounds, as for example, ammonium nitrate,
ammonium chloride or urea may be used in place of potassium
nitrate. When ammonium chloride is used a pH must be contin-
uously adjusted to neutrality to avoid excess culture acidity.
As indicated below, in certain of the examples the amount
of nitrogen was varied from that given above. In other examples,
culturing was continued in the nutrient medium until the nitro-
gen in the medium was used up.
11)4~Z~
The term polysaccharide i5 used herein to mean a material
which is an ethanol precipitable, non-dialyzable, anthrone sugar
reacting material. In the examples, viscosity is measured by a
calibrated viscometer sold by Cannon Fenske and results are
expressed in centistokes. Whole culture viscosity is
approximately equal to the viscosity of centrifuged cell free
supernatent. Culture growth is measured at a wave length of 690
nm by a colorimeter manufactured by Bausch & Lomb under the
trademark Spectronic 20. Cell number and culture dry weight are
linear functions of optical density up to an optical density of
1. Dry weight is measured directly or estimated from the
relationship g/L = O.D. 690 which has been empirically derived,
wherein g is grams of cell matter, L is volume of algae and
medium in liters and optical density (O.D.) is measured at 690
nanometers.
When cells from an exponentially growing culture of
Chlamydomonas mexicana are isolated by centrifugation, their
carbohydrate content is around 35% of cellular dry weight.
Because of the fragility of these cells and the tendency for
capsular materials to slough off it is difficult to strictly
classify the carbohydrates as extracellular. What can be said
in general is that when cultures reach the end of exponential
growth about 50% of the total culture carbohydrate is soluble
and cell free (isolated by centrifugation). Twenty-four hours
later up to 90~ of whole culture carbohydrate is soluble and
cell free. Approximately 90% of soluble cell free carbohydrate
is precipitable by 2 volumes of ethanol. Unless otherwise
stated all carbohydrate determinations are whole culture
determinations.
Cellular nitrogen content is determined by the Kjeldahl
nitrogen method (Standard Methods, 13th Edition, 1971) in
--10--
104~Z2
vegetative or early zygote cultures. In older cultures cellular
nitrogen is computed based on the culture dry weight and initial
nitrate nitrogen level. Nitrate nitrogen is also determined by
Standard Methods (13th ~dition, 1971).
Example I
The green algal species Chlamydomonas mexicana, strain
#729, (Indiana University Culture Collection) was placed asep-
tically in 250 ml Erlenmyer flasks containing 50 ml of sterile
medium prepared as in Table I and incub~ted until the optical
density at 690 nm reached 0.5 (250 mg/L dry weight). The entire
culture was transferred to a 1 L Erlenmyer flask containing 500
ml of the same medium. This medium provided for continued
exponential growth for about one more day at which point
cellular nitrogen was at about 5% by weight as will be shown
herein below. The culture was bubbled constantly with 5% CO2 in
air at a light intensity of 500 foot candles. Lighting was
continuous for the duration of the experiment. The results of
culturing as described above over a 10 day period are shown in
Figures 1-3 and Table II.
Figure 1 shows a plot of culture growth, carbohydrate,
viscosity and flocculation value as a function of time~ Floc-
culation value in this example is a measure of the minimum
amount of active material required to produce the first visible
aggregation of clay particles and provides a rating of active
materials by comparing the lowest dosage required to produce
visible aggregation. A suspension of kaolin clay is prepared
with an average particle diameter of non-flocculating kaolin
clay being 3.2 microns. The average particle diameter of kaolin
at the smallest dosage which causes visible aggregation is 20
microns. The inverse of the volume required to cause a particle
~04~)1Z2
diameter of 20 microns, is referred to 1 and is used as a
measure of flocculation value. An initial substrate nitrogen
level of 25 mg/l was chosen after taking into account such
growth regulating factors as light intensity, CO2 and
temperature. This level of substrate nitrogen resulted in the
production of 250 mg of algal dry weight having a nitrogen
content of 10%. This dry weight corresponds to an optical
density (690 nm) of 0.5 which occurred during the early expo-
nential growth phase. As can be seen in Table II and Figures 1
and 3, culturing was continued for about 10 days.
TABLE II
-
~rowth O.D. Cellular Dry
Day (690 nm) Cells/mlWeight ~/L ~N
0 .045 1 x 106 .022 10
1 .168 2.5 x 106.0840 10
2 1.08 1.9 x 107.540 4.6
3 1.25 2.5 x 107.620 4.0
7 1.29 2.6 x 107.640 3.9
1.58 3.1 x 107.790 3.2
All measurements are based on whvle culture determinations
except for cellular dry weight which is obtained from centri-
fuged exponential cells. Cellular dry weight from cultures in
the post exponential phase is estimated from the optical density
according to the empirical relationship mentioned above.
The data presented in Figure 1 and Table II shows that `
culture carbohydrate, flocculation value and viscosity do not
parallel culture growth but increase after growth ceases. By
the end of exponential growth (OD 690 = 1.0) cellular nitrogen
has fallen to 5% of culture dry weight. This post exponential
phase (starting with day 2 in the illustrative example) is the
flocculant production phase.
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The relationship between flocculation value, carbohydrate
content and cellular nitrogen is illustrated in Figure ~. ~s
can be seen in Figure 2, substantially no Elocculant is produced
until cellular nitrogen reaches about 5~ of dry weight. The
flocculant value and carbohydrate content are seen to rise in an
exponential fashion as the cellular nitrogen content falls below
5~.
Figure 3 shows plots of carbohydrate per cell versus time.
Tt is apparent that the amount of carbohydrate (i.e.,
flocculant) per cell begins to increase as cell multiplication
slows (day 2) and reaches a maximum 5 days later. Thus, the
phase of rapid cellular multiplication is clearly separated from
the phase of maximum cellular polysaccharide production. The
slope of the flocculant production curve represents the rate of
flocculant production. During rapid cell multiplication this
rate is less than 1/3 the rate found during stationary phase of
growth.
Example II
For comparison purposes, two 8" diameter plexiglass cylin-
ders containing 34 liters of nutrient medium were inoculatedwith Chlamydomonas mexlcana from intermediate flasks to produce
a starting O.D. of 0.1 at 690 nm (50 mg/L). Five percent of CO2
in air was bubbled through air stones positioned on the bottom
of the cylinders. Culture A contained about 23 mg/L nitrate
nitrogen and Culture B, about 47 mg/L of nitrate nitrogen. The
nutrient medium composition was otherwise identical to the
medium of Table I. The nitrogen in the nutrient medium
composition of Culture B was two times more concentrated than
Culture A. Culture growth as measured by optical density and
viscosity are shown in Figure 4. Culture A showed the
characteristic phase of exponential growth followed by the phase
of flocculant production as indicated by the increase in
viscosity. Culture B, however, showed only the phase of
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exponential growth. Culture A reached a cellular nitrogen
content of 5% of dry weight by 50 hours whereas Culture B
remained at 10% cellular nitrogen for over 100 hours.
Example III
This example illustrates use of one form of culturing sys-
tem well suited for carrying out the invention and shows how
the manipulation of culture nitrogen levels can be used to pro-
duce algal flocculants in a large scale semi-continuous culturin~
process. The flocculant produced from such a system lends it-
self for direct use although it may be further concentratedif desired.
The culture vessels employed, one of which is shown at 4 in
Figure 5b, consisted of two rectangular glass tanks having a ca-
pacity of 160 L each. The culture tanks 4 were continuously il-
luminated with fluorescent lights on all four sides from lamps 1,
2 and 5 and a fourth lamp which is not shown for clarity of il-
lustration. The culture medium was continuously bubbled with 5%
C2 from cylinder 11, via valve 10 and flowmeter 6. Air is pro-
vided by means of compressor 8, valve 9 and flowmeter 7. Both air
and CO2 enter the tank through air stones 13, one of which is also
shown in Figure 5a. Temperature was maintained at 25C by use of
a circulation system including pump 14, heat exchanger 15 and a
fan 12. The first culture tank I is operated as the vegetative
growth chamber whereas the second tank II i5 operated as the floc-
culant production chamber. At initial start-up, the first tank is
inoculated with culture (Chlam~omonas mexicana) in 80 liters of
the aqueous medium of Table I which contains 25 Mg/L N. This lev-
el of nitrogen results in an algae density of 250 mg/L of 10~ nitro-
gen
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by weight vegetative cells ~O.D. 690 = Q.5~. When the first tank
reaches this density, a portion of its con~ents, pre~erably about
one-half of its volume, is transferred to the second tank. Tank I
then receives nutrient medium (25 mg/L N~ to original volume. Tank
II receives an equal volume of water. Both tanks are permitted to
grow to a final optical density of about 0.5, which corresponds to
cells having a nitrogen content of 10% in Tank I, and less than
5% in Tank II.
Flocculant accumulates in Tank II. The contents may be
lo removed and used as is or after the cells are separated as by
centrifugation, as desired. The system is schematically depicted
by the flow chart of Figure 6 and is capable of operation as
described every 2-3 days.
As intimated from Example I, another mode of carrying out
the invention involves culturing under conditions for producing
flocculants in the same vessel in which vegetative growth occurs.
We have achieved the production o-f extracellular polymers -from
Chlamydomonas mexicana out of doors in shallow 12 foot diameter
pools provided with CO2 enriched air. It is preferred that the
contents of the pools be vigorously mixed to prevent sedimentation
of the algae. Viscosities similar to those obtained in Example III
can be obtained under proper light and temperature conditions when
nutrient levels are controlled as taught herein.
It is important that adequate levels of other nutrients
besides nitrogen be maintained in the nutrient solution to support
growth. In carrying out the invention, phosphorous levels must be
high enough to satis~y exponential growth requirements. If cellu-
lar phosphorus levels are too low, then growth may be limited by
lack of phosphorus with the result that cellular nitrogen never
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2Z
falls below the level required for appreciable flocculant produc-
~ion. Phosphorus requirements for healthy cell growth are
generally understood by those in the art. If uncertainty exists
as to a particular species, a few experimental runs will establish
adequate phosphorus levels.
The substances in Chlamydomonas mexicana cultures
responsible for viscosity, anthrone sugar reaction as well as
flocculant activity are ethanol precipitable, non-dialyzable and
therefore are considered to be high molecular weight polymers.
Example IV
A mesophilic strain of Chlorella pyrenoidosa (#343)
was obtained from the University of Indiana algae collection.
Cultures were grown in Fernbach flasks under continuous aeration
and white light of 400 fc intensity. A nutrient medium of the
following composition was employed.
Compound gtL Micronutrients ~
CaC12-H2O .016 H3BO3 2.86
MgSO4-7H20 .250 Mncl2-4H2o 1.81
KNO3 ~nSO4~H2O .22
K2HPO4 .030 MoO3 .017
NaHCO3 .020 CUso~t5H2o .079
Iron (EDTA .005 CC12-6H2 .041
Micronutrients 1 ml/L
Distilled Water 1 L
~ locculant production by Chlorella pyrenoidosa was
studied under two conditions. Culture I had sufficient nitrogen
and phosphorous to produce a cellular nitrogen and phosphorous
content of 5.1 and 0.58% respectively by day 11. Culture II con-
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3L0~ 2
tained the same nitrogen level as Culture I but was phosphorous
deficien~. Figure 7 shows a plot of flocculation values versus
time.
In assaying flocculation, the following procedure was
followed. Kaolin clay stock and diluent solutions were prepared
as indicated:
Kaolin Stock 0.1 g/l MgSO4-7H2O
a . 1 g/l NaCl
0.1 NaHCO3
lo 0.15 g/l CaCL2-2H20
0.2 g/l Kaolin ~Fisher)
pH adjusted to 7 with HCl
Stock stirred two days before use
Diluent 0.1 g/l MgSO4-7H2o
0.1 g/l NaCl
0.1 g/l NaHCO3
0.15 g/l ~aC12-2H2O
pH 8.6
Iron Stock 1.7 g/l FeC12-4H2O
Flocculations were carried out in 12 test beakers
containing 28 ml of kaolin suspension each. The suspension was
made by adding 240 ml Kaolin Stock and 160 ml diluent. 0.04 ml
of Iron Stock was added and the mixture stirred for 30 minutes
~the O.D. = 0.250, pH = 7.6). Twenty-eight ml of the kaolin
600
mixture was dispensed into 50 ml beakers. Flocculant was added to
each beaker with 30 seconds of rapid stirring in 30 second inter-
vals. After addition, beakers were stirred for one hour at 30 rpm.
In 30 second intervals, the beakers were gently agi~ated and 6 ml
poured into cuvettes. After 30 minutes of settling, the O.D. ~at
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600 nanometers) o each cuvette was read at 30 second intervals.
One control was run with each 6 test beakers. An O.D.600 of 0.250
corresponds to a kaolin concentration of 120 g/ml. The 28 ml
assay volume therefor contained 3360 g kaolin. Control cuvettes
usually had an O.D.600 = 0.180, e~uivalent to 2420 g kaolin. A
sixty percent reduction of relative O.D., therefore, corresponded
to a flocculation of 1450 g kaolin.
Although in Figure 7 both cultures had identical growth
kinetics only Culture I showed significant flocculant activity.
The maximum flocculation value (Culture I) occured between about
day 11 and day 15. At day 11 Culture I and II had a similar
cellular nitrogen level (5.1 versus 4.3% respectively), but
differed significantly in cellular phosphorous (0.58 versus 0.21%
respectively). m e unusually low phosphorous level in Culture II
has been found to correlate with the absence of flocculant
activity. Since Culture II was found to contain two to three
times greater dia~yzable (small molecular weight) saccharides
than Culture I, it is theorized that low cellular phophorous
levels cause depolymerication of polysaccharides or inhibit their
formation.
Under certain circumstances, we have observed that
phosphorous deficient Chlorella cells can be made to produce
flocculants. As is recognized in the art, an actively growing
population of Chlorella consists primarily of D or "dark'l cells
which are characterized by being small but with high
photosynthetic and low respiratory activity. When D-cells are
transferred to a medium deficient in nitrogen they can undergo a
transition to L cells which in turn can undergo division. These L
or "light" cells are somwhat larger than D-cells having low photo-
synthetic and high respiratory activity. By way of comparisonthese cells have an average weight of 6 x 10 11 gms/cell as
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A~;.
~)4a)~Z~
compared ~ith the ~eight o dar~ cells which averagea 2 x lQ
gms/cell. At low phosph~rus levels, i.e. below a~ou~ .3~ of dry
weight, L-cells ha~e ~een observed to sometimes give rise to
flocculant activity. However, we have found that from the stand-
poin~ of maximizing productivity, phosphorous levels of a~out
about .3% dry weight and preferably above about .5% dry weight
should be maintained.
Example V
In order to illustrate the effectiveness of the
flocculants of the invention, as compared with a polystyrene
sulfonate flocculating agent, comparative filtration rates through
filter cakes were measured by the following procedure, which con-
sists of two separate steps:
1) dispersal of the flocculant into the kaolin suspension;
2) filtration of the liquid through the clay.
Step 1. A two blade marine type propeller was used to
mechanically mix -flocculant with the colloidal clay kaolin. Con-
stant mixing speed was obtained by attaching a tachometer to the
stirring shaft. Time and speed of mixing was held constant for
each dose of flocculant.
Step 2. ~locculant materials prepared as in E~ample I or
polystyrene sulfonate were added dropwise to a mechanically stirred
kaolin suspension at a clay concentration o-f 1 g/100 ml. The
flocculant solutions ranged in concentration from 0.1 g/L ~0.01%)
to 1 g/L ~0.1%). The final volumes of flocculant-clay suspensions
were held constant.
Measured parameters for the mixing step include:
Ms.= speed in RPM of shaft (constant)
Mt = total time in mixing (constant)
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Dp = dose o~ flocculant in ml ~independent variable)
Cp = concentration of flocculant solution ~con~tant~
ml o~ ~tock kaolin suspension (constant)
Ck = concentration o-f stock kaolin suspension in mg/l
(constant)
Vm = final volume after mixing (constant)
The filtration apparatus consisted of Buchner funnels ~7 mm diameter
by 50 mm height with stopcocks in the stem to provide contTolled
time. Glass filter paper of less than 2~Upore size was used.
The collecting ~rlenmyer flasks were connected to a manifold
pretested to draw equal vacuum from each port. A small vacuum
pump and a mercury manometer were used to produce and monitor
the vacuum.
The mixed flocculant - kaolin suspension was transferred
from the beakers to the funnels which were maintained under a
vacuum to seal the filter paper to the funnel bottom. Stopcocks
were closed and the suspension allowed to settle. Subsidence and
clarity of the superna~ent were noted. The suspensions were
filtered and then re-filtered. The volume collected upon re-
filtration with constant vacuum for a constant length of timewas measured. A measure of filtration was taken as the ratio
of the re~iltration rate of the control to the refiltration rate
of i:
f = refiltration rate of control = V
refiltration rate i ~C/ft
i = flocculant-kaolin suspension Fi/~t
Measured parameters were:
P = vacuum in inches H (constant)
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*Vm = initial Eiltration volume (constant)
fT = refiltration time (constant)
VF = volume collected after refiltration
~dependen~ variable)
VF = volume collected after refiltration control
(constant)
Figure 8 is a plot of Eiltration rate versus parts polymer to
million parts of kaolin. Eight million molecular weight sodium
polystyrene sulfonate and an algal flocculant from Chlamydomonas
mexicana cultured as described herein were compared. Both
polymers or flocculant react with the clay at a polymer to clay
ratio of 1 to 5000.
Example VI
As indicated above, algal flocculants are an effective aid
in the coagulation of solids in waste water. Specifically, we
have found that the use of Chlamydomonas mexicana algal
flocculant in conjunction with lime results in the production of
larger, denser and therefor faster settling flocs than is the
case with lime alone. The amount of lime required appears to be
less as a result of the use of the algal flocculant. In one
example, screened influent from the Deer Island Sewage Treatment
Plant in Boston, Massachusetts was flocculated with lime and
lime/algae flocculant usin~ the jar test flocculation procedure
described in Example IV. The flocculants which were prepared in
accordance with Example I, were added in a rapid mix, slow
addition fashion and stirred for one minute in beakers. The
beakers were then placed on a Phipps and Bird Stirrer at 6 rpm for
five minutes and a final 20 rpm for thirty minutes. At the end of
thirty minutes the settling time for subsidence to clear 95% of
the supernatent volume was recorded.
* When Vm exceeded 100 ml the difference was discarded from
supernatent.
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Lime was applied as a 10 g/L slurry and in sufficient
quantity to reach the desired pH. One ml of Chlamydomonas
mexicana flocculant was prepared in accordance with ~he technique
of example III (5 9/~, viscosity - 30 centistokes) and was added
to each 1 L of sewage.
The effect of algae flocculant and lime compared to lime
alone is seen in Table III. Lime doses necessary to produce pH
10, 10.5 and 11 were compared. The lime and algae flocculant
caused the flocculated sewage to settle 10 times faster than lime
alone at pH 10 and 10.5. No difference between the two are
observed at pH 11 although lime and algal flocculant produced a
larger floc size.
TABLE III
Settling Time Ratios (Minutes)
(Lime/lime & Algal Flocculant)
10 . O 10/1
10 . ~ 10/1
11. 0 1/1
Examples VII and VIII
Polymeric substances such as polyacrylamides, bacterial
polysaccharides and alginic acid improve soil structure by
forming water stable aggregates which in turn improve water flow
and air penetration into soil. The following examples are
illustrative of the soil conditioning properties of Chlamydomonas
mexicana on western calcareous soils.
22-
~ xample VII illustrates the water st~ab.le a$gre~ate
formation resulting ~hen Chlamydomonas flocculating agent is mixed
with soils at relatively high dosages. Example VII illustrates the
effect of a relatively low dose of Chlamyd~monas flocculating
agent on soils under field conditions.
In carrying out Example VII, a dry calcareous soil
~20% clay, 53% silt and 27% sand) was passed through a l mm sieve
to remove particles greater than 1 mm. Fifty gram portions of
sieved soil were mixed with algae cultures having flocculant
lo activity. Culture dry weight to soil dry weight ratios of 1:1000,
1:2500, and 1:5000 were made. A control consisting of algae free
culture medium was also mixed with soil. Mixing was carried out
for five minutes with a spatula. The samples were dried at 50C
to a soil moisture of around 10%. This moist soil was passed
through a 2 mm sieve and the artificial aggregates formed greater
than 1 mm were used for the aggregate stability study.
Aggregates less than 2 mm and larger than 1 mm in size
were dried at room temperature and wet sieved by direct atmospheric
immersion for ten minutes. (Method of Soil Analysis Agronomy
Monograph No. 9, Part I, pp. 511-519.) The nest of sieves consisted
of 1, .5, .25, .1 and .05 mm sieves. The dry weight soil retained
on top of each sieve was determined. The results are listed below:
TABLE IV
Chlamydomonas
Flocculant to % Retained
Soil Ratio (w/w} ~ 1 mm ~p.25 mm ~1 ~n
~.1% mixture) 1:1000 96 74 60
(.04% mixture) 1:2500 96 84 41
(.02% mixture) 1:5000 58 24 11
control 37 25 13
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The results show that nearly five times more soil is retained on
the 1 mm sieve at a dose of 0.1% of flocculant in soil than is
retained by the 1 mm sieve of the control of untreated soil. At
the intermediate dose of flocculant to soil of 1:2500, three
times more water stable aggregates are retained on the 1 mm sieve
when compared to the control. Sixty-four percent more soil is
retained on all sieves greater than 0.1 in the low flocculant to
soil dose compared to the untreated soil.
In Example VIII, triplicate plots of 81 ft2 each were given
the following treatment:
(a) control (water)
(b) Chlamydomonas flocculant 12.5 lg/acre
(c) Chlamydomonas flocculant 50 lb/acre
(d) Chlamydomonas flocculant 200 lb/acre
Flocculant was prepared as in Example I. Quantities are
measured in terms of pounds of dry wei~ht.
Sixty-four gallons of each treatment product was applied
per plot. Each plot was then rototilled to a depth of 6" after
soil moisture reache~ a tillable level. Soil structure changes
were assessed by the following measurements: wet sieving,
infiltration and penetration resistance.
Figure 9 shows a plot of each of the three physical measure-
ments versus does of algal flocculant applied. All values are
the mean of three replications. In the horizontal scale, (dose)
1/8 was chosen so that the data fit a straight line, for
illustrative purposes.
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z
Figure 9a shows the effect of varying levels o~ flocculant
on water infiltration rate, measured ~or 8 hours at a one-inch
head. The results show a 100% increase in infiltration rate o~
treated plots versus untreated control plots. A dose response is
also evident in the positive slope of the curve. The data is
significant at the 75% level.
The effect of algal flocculant on penetration resistance
is shown in Figure 9b. This parameter is a measure of the force
required to push a conical-tipped probe four inches into the
soil. Greater than 50% decrease in resistance occurred as a
result of treatment with flocculant. A dose response is
indicated by the slope of the curve. The data is significant at
the 90% level.
Results shown in Figure 9c show an increase in mean particle
size of flocculant treated soil samples as measured by wet
sieving. A 15% increase in mean particle size occurred after
treatment with 200 lb/acre. More significantly, a dose response
to flocculant is evident by the positive slope of the curve. The
data is significant at the 97.5% level.
Although the invention has general applicability to the
production of flocculating agents from algae, it is of particular
utility in the production of flocculating agents from uni-
cellular, green and non-nitrogen fixing blue-green species which
are normal inhabitants of soil and ~resh water. The selection of
a species suitable for the purposes of the invention is first
based on an evaluation of its potential for mass culture. Growth
rate should preferably be a minimum of one doubling per day. ~nce
a selection of a promising species is made, culturing is carried
out under cvnditions in which nitrogen is restricted so that the
cell becomes nitrogen deficient, i.e. below about 5% by weight of
cellular nitrogen with other nutrients being favorable for
growth. During this process, the production of extracellular
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flocculant is monitored as described herein and the yreatest
flocculant producers selected.
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