Cyanobacterium-produced bioemulsifier composition and solution thereof

J-1 which is a strain of cyanobacteria is used to form and excrete a material useful as an emulsifying agent for forming emulsions of hydrocarbons and oils in liquids such as water. Method of separating and culturing the cyanobacteria under conditions necessary to achieve a maximum formation and excretion of the emulsifying agent into solution. Method of purifying and separating excreted as well as intracellular material from cyanobacteria. Method of removing stains with a material excreted by cyanobacteria, and particularly strain J-1. Method of effecting the secondary recovery of petroleum through the use of a material excreted by cyanobacteria, and particularly strain J-1. Extracellular polymeric material which is greater than 200,000 Daltons in molecular weight, and contains sugar, fatty acid, and protein moieties, and amide, carboxylic and amino groups.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The invention relates to a method of separating bacteria from a liquid, and 
to a method for culturing the bacteria to produce a polymer useful as an 
emulsifying agent. The invention further relates to the polymeric 
substance produced, and to methods of using the polymeric substance. 
2. Description of Pertinent Materials 
Many situations exist in which it is desirable to utilize an emulsifying 
agent to disperse one substance in another. More particularly, an 
emulsifying agent is often used to disperse a hydrocarbon material or oil 
into another medium, such as water. 
The number of situations in which it is desirable to utilize an emulsifying 
agent to disperse various types of oils or hydrocarbons are many and 
varied. For example, emulsifying agents may be used in the dispersal of 
oils in various types of food preparations, in the secondary recovery of 
petroleum, and as a stain remover. 
A large portion of valuable petroleum is not recovered from wells through 
pumping techniques, because the oil or other material sticks to the 
surface of dirt particles. An emulsifying agent can loosen the trapped 
petroleum, which can then be recovered. Emulsifying agents are also useful 
in the cleaning of residual oils from oil tankers, in the preparation of 
alcohol-oil mixtures for use as fuels, and in the dispersion of oil spills 
or slicks on bodies of water. 
It is currently customary to use manufactured chemical preparations as 
emulsifying agents. Such materials are often quite expensive. 
It would, therefore, be desirable to use as emulsifying agents materials 
which could be readily and inexpensively produced or secured from natural 
sources. 
SUMMARY OF THE INVENTION 
It is an object of the invention to provide a process for the production of 
a novel emulsifying agent, useful in the emulsification of hydrocarbons 
and oils. 
The objective of the invention is achieved through the use of cellular 
products of a benthic filamentous cyanobacterium of Phormidium genus in 
the family Oscillatoriaceae. This strain has been found to exist in a 
portion of the Jordan river, where it was noticed that the water seemed to 
be clarified of its particulates, as discussed in patent application Ser. 
No. 634,535, filed July 26, 1984, and now U.S. Pat. No. 4,649,110, of 
which this application is a continuation-in-part. One particular bacterial 
strain of Phormidium useful in the invention has been denominated as J-1. 
J-1, is, in effect, a definite strain of an undefined species of the 
Phormidium genus. A specimen of this organism has been deposited with the 
American Type Culture Collection in Rockville, Md., and has been assigned 
ATCC No. 39161. This strain has a generation time of 24-48 hours, and 
under the carefully controlled conditions of the invention, the organism 
produces and excretes a polymeric bioemulsifying material which is useful 
in achieving the objectives of the invention. The polymeric bioemulsifier 
can also be secured from the bacterial cells themselves, by rupturing the 
cells and then isolating the bioemulsifying agent. 
The organisms of the invention are found naturally in certain fresh water 
bodies. These organisms can be collected and removed from the water for 
culturing, according to the inventive technique, by adding nonpolar 
hydrocarbons into the water containing the bacteria, and agitating the 
water to disperse the hydrocarbon into globules, as described in U.S. 
application Ser. No. 634,535, now U.S. Pat. No. 4,649,110 discussed above. 
Because benthic bacteria (including members of the Phormidium genus) are 
hydrophobic, the bacteria become attached to the dispersed globules of 
hydrocarbon. The hydrocarbon is collected as it floats to the water 
surface, and the bacteria can then be separated from the hydrocarbon and 
grown under laboratory conditions. 
Once the hydrocarbons have been separated according to the inventive 
technique, separated cyanobacteria are ready for treatment according to 
another aspect of the invention. 
Under specially controlled and tailored growth conditions, using properly 
selected media, and under defined physiological conditions, the 
cyanobacteria of the invention can be made to produce and excrete 
effective amounts of a polymeric substance useful as an emulsifying agent, 
and referred to in this application as a emulcyan. 
According to the invention, the bacteria produce emulcyan towards the end 
of an exponential growth phase and excrete the active, 
high-molecular-weight substance into the external milieu during a 
subsequent stationary phase. This substance is extremely effective as an 
emulsifying agent. 
For the exponential growth phase to occur, the microorganisms must be 
provided with an adequate amount of light, ideally ranging from 20 to 30 
.mu.E/m.sup.2 /sec.sup.-1. The temperature should range between about 
27.degree.-33.degree. C. The mineral requirements of the organisms are 
preferably met by using a Chu-11 medium which is made as described in the 
scientific article entitled "Hydrophobicity as an Adhesion Mechanism of 
Benthic Cyanobacteria" by Ali FATTOM and Moshe SHILO (Applied and 
Environmental Microbiology, January 1984, Vol 47, No. 1 pp.135-143. 
The bioemulsifier described in this invention has a molecular weight above 
200,000 Daltons. Chemical analysis indicates that it contains sugar, fatty 
acid and protein moieties. Enzymatic degradation of 70% of the protein 
content does not affect the emulsification activity. Infrared 
spectrophotometry indicates that the bioemulgent contains amide, 
carboxylic and amino groups. 
The bacteria are subjected to careful growth conditions so as to produce 
effective concentrations of the bioemulsifier. The bacteria are first 
subjected to conditions necessary to achieve an exponential growth 
condition as discussed in the parent application. This treatment is 
continued for approximately six days until the bacteria begin to produce 
increased levels of the bioemulsifier towards the end of the six day 
period. Exponential growth is then stopped and the bacteria are made to 
enter a resting phase by reducing the calcium ion concentration in the 
solution from about 35 mg/l to about 350 mg/liter. During this phase, 
effective levels of the polymer are excreted into the surrounding 
solution. 
Although the entire crude bacteria-containing solution may then be used as 
an emulsifying agent, according to one embodiment of the invention, the 
bioemulsifier is separated from the remainder of the solution. The 
separation may be performed by centrifuging the cell culture, such that a 
first pellet (or filtrate) is produced, along with the corresponding 
supernatant. Both the pellet and the supernatant are subjected to a series 
of treatment steps to provide maximum recovery. The first pellet formed as 
mentioned above will yield the cell-bound bioemulsifier, which had not yet 
been excreted by the bacteria, while the supernatant will yield the 
extracellular bioemulsifyer which had already been excreted into the 
culture medium before the original centrifugation. 
Likewise, as discussed in the parent application Ser. No. 634,535, now U.S. 
Pat. No. 4,649,110 the first pellet will also yield the cell-bound 
bioflocculant, while the supernatant will also yield the extracellular 
bioflocculant, when subjected to the series of treatment steps. In the 
above-mentioned parent application, a series of treatment steps is 
described for producing two pellets of bioflocculant. Subsequent study has 
shown that these two corresponding supernatants also contain the 
bioemulsifier of the present invention. This bioemulsifier can be 
separated from its solution by flash evaporation. 
Although the above technique is cell destructive, the invention likewise 
optionally includes techniques in which only the original supernatant is 
treated to remove the bioemulsifier, while the pellet containing the cells 
is reused by being subjected to another exponential growth phase.

DETAILED DESCRIPTION 
The bacteria of the invention have been found in nature and may be cultured 
either in the laboratory or in their natural environment to produce the 
inventive composition. 
When it is desired to obtain a concentrated sample of the inventive 
bacteria from their environment in bodies of water, the bacteria are 
separated from their natural environment and then may be cultured under 
controlled conditions. 
Separation may be performed by means of a filtration or by centrifugation 
separation techniques. 
According to a liquid-liquid separation technique of the invention, one or 
more non-polar water immiscible hydrocarbons such as xylene, heptane, 
octane, hexadecane, and mixtures thereof, are added to the 
bacteria-containing body of water and agitated to form globules. By virtue 
of the non-polar nature of the hydrocarbons, benthic bacteria (including 
some members of the Phormidium genus, particularly the J-1 strain) will be 
adsorbed onto the hydrocarbon globules by virtue of their hydrophobic 
nature. By dispersing the hydrocarbon into small globules, improved 
separation is achieved. This occurs by virtue of the improved 
liquid-liquid contact, providing a greater effective globule surface area. 
Depending upon the nature of the environment, various agitation and/or 
mixing techniques may be used to accomplish this purpose. 
After sufficient agitation, the hydrocarbon material with the J-1 adhering 
thereto is separated from the liquid. The hydrocarbon globules, such as 
heptane, may be collected by centrifugation or filtration and then 
separated. The bacteria are then separated from the hydrocarbon phase by 
any one of a number of standard techniques. For example, small scale 
separation may be achieved by filtration of the bacteria cells on glass 
microfiber filters. On a large scale other filtration techniques may be 
used. 
It should be noted that other hydrophobic bacteria, other than strain J-1, 
which may be present in the original liquid environment, may also 
unavoidably be removed by means of the liquid-liquid separation technique 
referred to above, when the bacteria are found in an uncontrolled 
non-laboratory environment. When this is the case, the J-1 strain is 
preferably subsequently separated from the undesired bacteria when a pure 
culture is desired, through isolation of individual cells by methods known 
to those skilled in the art. 
Once the bacteria have been collected, and are in a medium where variables 
can be controlled, the bacteria may then be conditioned so as to produce 
effective amounts and concentrations of the emulsifying agent. 
The bioemulgent is preferably prepared in the following manner: 
Cyanobacteria of Phormidium genus (strain J-1) do not ordinarily produce 
sufficient quantities of the described emulcyan. The production of large 
amounts of the bioemulgent is dependent upon the physiological state of 
the organism. To achieve sufficient production, the organism must be 
allowed to proceed from 
an exponential growth phase, to a late phase of stationary growth. During 
this exponential growth phase the proper nutrients must be readily 
available. The nutrients are preferably provided by using a Chu-11 medium. 
Essential materials include nitrogen and phosphate as well as light and 
carbon dioxide from the atmosphere. The temperature range is preferably 
between 27.degree.-33.degree. C. 
As may be seen from FIG. 2, the exponential growth phase requires about 
twelve days, during which time the cell protein increases to about 
10.sup.2 .mu.g/ml. The production of cell-bound bioemulgent shows a 
regular steady increase almost from the outset. However, the production of 
extracellular bioemulgent does not show a sharp increase until about the 
eighth day of culture. 
As in the case of the bioflocculant of U.S. application Ser. No. 634,535, 
now U.S. Pat. No. 4,649,110 the amount of bioemulgent produced and 
excreted into the solution is markedly increased by lowering the available 
level of calcium ions in the culture medium, in order to subject the 
organisms to a state of relative deprivation of calcium, such that the 
organisms enter a dormant phase. Peak production of the bioflocculant of 
the above-mentioned parent application and the emulcyan of the present 
invention occurs at the same time. 
Calcium deprivation may be achieved either by limiting the amount of 
calcium added to, or present in the solution containing the microorganism, 
or by adding a chelating agent such as EDTA. 
As the emulcyan is partially free in the culture medium, it is possible to 
use the culture medium directly to emulsify the hydrocarbons and oils. 
However, it is most effective to separate the bioemulsifier from the 
Phormidium. This is done by a series of steps, whereupon the bioemulgent 
and the bioflocculant described in the parent application are separated 
from the bacteria. The separation of these substances is achieved as 
described below. 
As illustrated in FIG. 6, the cell culture is first subjected to 
centrifugation at 10,000 g for 10 minutes to produce an initial pellet and 
an initial supernatant. 
The pellet is resuspended in 0.02 M Tris buffer solution at a pH of 7.5, 
supplemented with 10 mM MgSO.sub.4. The cells in this solution are then 
subjected to disintegration by treatment with glass beads of 0.1 mm in 
diameter in a B. Braun Melsungen agitator maintained at power level 2 for 
two minutes. The resulting solution with the broken cells is then 
subjected to filtration on a GF/C fiberglass filter. Pronase (produced by 
the Sigma Company) is then added to the filtrate in the amount of 100 
.mu.g/ml of filtrate. (Pronase is a non-specific proteolytic enzyme formed 
by Steptomyces griseus.) This mixture is incubated at 37.degree. C. for 
one hour. Ethanol is then added at 66% final concentration to effect 
precipitation, and the mixture is chilled in ice water for one hour, after 
which it is subjected to centrifugation at 10,000 g for 10 minutes. 
As a result, a second supernatant and a second pellet are produced. The 
pellet is treated as described in the parent application to isolate the 
cell-bound bioflocculant, while the supernatant is subjected to 
evaporation in a flash evaporator at 45.degree. C., in a vacuum, to 
isolate the cell-bound bioemulsifier. 
The initial supernatant produced by the original centrifugation of the cell 
culture described above is subjected to volume reduction with a flash 
evaporator at 45.degree. C. in a vacuum. The concentrated supernatant is 
then subjected to Pronase treatment at a concentration of 100 .mu.g/ml at 
37.degree. C. for one hour, and subjected to dialysis for approximately 8 
hours against distilled water, whereupon ethanol is added, to a final 
concentration of 66%, and the mixture is chilled in ice water for one 
hour. The addition of ethanol causes precipitation of extracellular 
bioflocculant. The solution is then subjected to centrifugation at 10,000 
g for 10 minutes, to produce a supernatant and pellet. The pellet 
constitutes one part of the bioflocculant discused in the parent 
application, as mentioned above. The supernatant is subjected to 
evaporation in a flash evaporator at 45.degree. C. in a vacuum to isolate 
the extracellular bioemulgent. 
Clearly, the treatment of the initial pellet resulting from the original 
centrifugation of the cell culture is cell destructive, and requires that 
a fresh batch of microorganism be provided if the production of the 
bioemulgent is to be repeated. However, where it is desired that the 
microorganisms be reused, the cells clearly should not be destroyed. Cells 
can be taken from the dormant phase and reconditioned by the proper 
treatment and can once again undergo exponential growth, whereby the 
process can be repeated. If it is decided to destroy the cells, the 
destroyed cells can be used as animal feed. 
It should be noted that the technique illustrated in FIG. 6 provides a 
method for removing any intracellular bioemulsifier remaining within the 
microorganisms. Although it is preferable to first subject the 
microorganisms containing increased levels of emulcyan to a stationary 
phase which results in the excretion of the bioemulsifier, if, for some 
reason, the microorganisms are not subjected to the stationary phase, the 
cells containing increased levels of 
emulcyan may simply be destroyed to recover the intracellular emulcyan. 
The bioemulsifier of the invention has a molecular weight above 200,000 
Daltons. Chemical analysis indicates that it contains sugar, fatty acid 
and protein moieties. Enzymatic degradation of 70% of the protein content 
does not affect the emulsification activity. Infrared spectrophotometry 
indicates that the emulcyan contains amide, carboxylic and amino groups. 
FIG. 7 is a reproduction of the IR spectrum associated with the emulcyan, 
listing the functional groups whose presence is indicated. 
To determine the emulsification activity of the 
emulcyan, varying quantities of it were mixed into a 0.02 M Tris buffer at 
a pH of 7.5, supplemented with 10 mM MgSO.sub.4. The final volume of each 
mixture was 7.5 ml in 125 ml Erlenmeyer flasks. Following this, 0.1 ml of 
a mixture of hexadecane and 2-methylnaphthalene (1 to 1 volume to volume) 
was added, and each flask was shaken for 1 hour at 150 strokes per minute 
at room temperature. The suspension was then read in a Klett-Summerson 
colorimeter at 540 nm. One unit of emulsification activity gave a 13.3 
Klett unit increase in the optical density reading. 
As seen from FIG. 5, emulsification activity is temperature dependant, and 
is shown to be at 100% of maximal value at a temperature of approximately 
26.degree. C. Temperatures lower or higher than this ideal temperature 
cause the bioemulsifier to exhibit a lower emulsification activity. For 
example, at 15.degree. C. the emulsification acitivity is 45% of maximal 
value. At 55.degree. C., the emulsification activity is 30% of maximal 
value. The experimental procedure used to generate the data for FIG. 5 is 
the same as that utilized for FIG. 3, discussed below, except that the 
mixtures were shaken at the appropriate temperatures. 
To test the effect of pH on the emulsification activity of the emulcyan (as 
shown in FIG. 3) 65 g/ml of the emulcyan were dissolved in a 0.02 M Tris 
buffer, supplemented with 10 mM MgSO.sub.4. To this solution, 0.1 ml of a 
mixture of hexadecane and 2-methylnaphthalene (one to one, volume to 
volume) was added. The final volume was 7.5 ml in a 125 ml flask. NaOH or 
HCl were added to obtain the appropriate pH. The flasks were shaken 150 
strokes per minute at 26.degree. C. for 1 hour. The turbidity of the 
resulting emulsion was measured in a Klett-Summerson colorimeter at 540 
nm. 
As shown in FIG. 3, the emulsifying agent is effective over a wide range of 
pH values, and may preferably be used at a pH value of anywhere from about 
3.0 to 11.0. However, emulsification activity is pH dependent, and, as may 
be seen from FIG. 3, the emulcyan is most effective at a pH of about 5-9. 
The values shown in FIG. 3 are expressed as a percent of maximal value. 
The activity level of the emulcyan is dose dependent (see FIG. 1). However, 
there is no clear optimum concentration. As seen from FIG. 1, 
emulsification activity (in Klett units) shows a steady increase as the 
crude bioemulgent concentration increases. This is unlike the performance 
of the bioflocculant discussed in the above-mentioned parent application, 
where there is an optimum concentration, at which flocculation activity 
reaches a peak. 
In testing the emulsification activity of the bioemulgent, as shown in FIG. 
1, different concentrations were prepared from purified emulcyan in a 
0.02M Tris buffer with a pH of 7.5, supplemented with 10 mM of MgSO.sub.4. 
As illustrated in FIG. 4, the emulsification activity of the emulcyan (as a 
percentage of maximal value) is dependent upon cation concentration in the 
medium being treated. FIG. 4 indicates, that with Mg.sup.2+, optimal 
concentration is around 4.0 mM, whereas the optimal concentration for 
Na.sup.+ is reached at approximately 25 mM. Up to each of these two 
points, the presence of increasing concentrations of these two cations 
results in a regular and sharp increase in emulsification activity. After 
the emulsification activity reaches 100% of its maximal value, additional 
quantities of cation will only cause the emulsification activity to stay 
at 100% of maximal value. However, excess quantities of cations will not 
do any harm to emulsification activity. The buffer used in connection with 
the experiments related to FIG. 4 is the same as that utilized in 
connection with FIG. 3, with the exception that the buffer was 
supplemented with the appropriate concentration of cations. The data for 
FIG. 4 were generated under ideal conditions of temperature (26.degree. 
C.) and pH (7.5). 
FIG. 2 illustrates the production, as a function of time, of cell bound and 
extracellular bioemulsifier, as well as cellular protein, in cultures of 
Phormidium. Phormidium J-1 cells are innoculated into a Chu-11 medium and 
the culture is incubated in an illuminated shaker at 150 strokes per 
minute, at a light intensity of 20 .mu.E/m.sup.-2 /sec.sup.-1. Samples are 
then taken periodically for the determination of extracellular and cell 
bound emulsification activity. Cellular protein is determined by the Lowry 
method. 
It is believed that the advantages and improved results furnished by the 
method of the present invention are apparent from the foregoing 
descriptions of the preferred embodiment of the invention. Various changes 
and modifications may be made without departing from the spirit and scope 
of the invention as described in the claims that follow.