CA1040122A - Production of algal bio-polymers - Google Patents

Abstract

PRODUCTION OF ALGAL BIO-POLYMERS

ABSTRACT OF THE DISCLOSURE

Cultivation of algae to produce long chain polymers having flocculating properties is disclosed. Algae are cul-tivated in an aqueous nutrient medium until optimum culture densities are achieved and thereafter under conditions in which they become deficient in nitrogen thereby causing the cells to shift from a growth phase in which protein production predominates to a growth phase in which extracellular polymer production pre-dominates. An adequate supply of other nutrients as well as CO2 and light are maintained in the culture medium during the latter phase to insure that a growth limitation is caused solely by a deficiency in nitrogen. The algae produce high molecular weight polymers exhibiting strong flocculating activity.

Description

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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3L~4~22 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-~3 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 -4a-;~

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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 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.

~ 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.

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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 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.

~4~122 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 ~Q~ 2;~
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 z~

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 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-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 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 ~4~1Z2 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 A~;.

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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) ~i~94~ZZ
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) *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.

~ 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 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.

~.~.

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 flocculant is monitored as described herein and the yreatest flocculant producers selected.

Claims (14)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A method of producing biopolymers exhibiting floccu-lating activity comprising; cultivating algae in the presence of light and carbon dioxide in a nutrient medium suitable 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.

2. A method according to claim 1 wherein the algae are selected from the group comprising unicellular green and non-nitrogen fixing blue-green algae.

3. A method according to claim 2 wherein the algae is of the genus Chlorella.

4. A method according to claim 3, further comprising the step of supplying the medium with sufficient phosphorus to maintain cellular phosphorus above about .5%.

5. A method according to claim 2 wherein the algae is of the genus Chlamydomonas.

6. The method according to claim 5 wherein the algae is of the species Chlamydomonas mexicana.

7. A method according to claim 1 wherein the exposure to light is continuous when the algae are grown in the nitrogen restricted medium.

8. A method of producing algal biopolymers having floc-culating activity comprising, cultivating an algae in an aqueous nitrogen rich nutrient medium in the presence of light and carbon dioxide until the culture reaches the desired density, thereafter continuing the cultivation until the cellular nitro-gen is below about 5%, and thereafter withdrawing the flocculant active polymers.

9. A method of producing polymeric material having floc-culating activity from algae, 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, thereafter 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 nitrogen content to below about 5% by weight and maintaining light, CO2 and plant nutrients other than nitrogen in quantities which are not growth limiting during the second period of time.

10. 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 CO2 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 transferred 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.

11. A method according to claim 10 wherein the second tank is substantially continuously exposed to light during the culturing step.

12. A method according to claim 10 wherein the algae is selected from the genus Chlamydomonas.

13. A method according to claim 12 wherein the algae is the species Chlamydomonas mexicana.

14. A method of coagulating solids in waste water which comprises adding lime and algal flocculant to the waste water.