Note: Descriptions are shown in the official language in which they were submitted.
~71~SO (5792)
MICROBIAL ENHANCEMENT OF POLYMER VISCOSITY
BACKGROUND OF THE INVENTION
Field of the Invention
This invention relates to the action of microor~anisms
on polymers contained in solution. More particularly, the
invention concerns a process for increasing the viscosity of a
polymer solution through the use of microorganisms.
: .
Discussion of the Art
For the purposes of this patent, all polymers can be
divided into two classes: biopolymers and synthetic polymers.
~:.
~` Biopolymers are polymers produced at least in part by the
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~ ~ action of biological processes, while synthetic polymers are
:
; produced by chemical processes.
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Biopolymers can be~obtained from a variety of natural
;~ plant sources, includin~ tree exudates, seed e~tracts, seaweed,
~starches, and cellulose derivatives. Some biopolymees, such as
:: ~ :
~ certain polysaccharides can be produced by microorganisms.
.
A well-known example of a microbial biopolymer is
xanthan gum, which is an anionic heteropolysaccharide made
exocellul~rly from carbohydrate substances by organisms of the
; genus Xanthomonas, such as X. campestris and X. be~oniae.
~; ; Xanthan and several other microbial biopolymers, such as the
fung~l product scleroglucan sold by the Pillsbury Company under
~, ,
the trade name "Polytran" and a polysaccharide ob~ained from
the~soil bacteria Erwin_ marketed under the trade name
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"Zanflo", are available in commercial quantities for
applications including inks and coatings, cosmetics, ceramics,
paint thickeners, drilling muds, pharmaceuticals, and foods.
Some microbial biopolymers have the ability to function as
surfactants, such as surfactin produced by Bacillus_subtilis.
These surfactants are useful as flocculating agents,
emulsi~iers, demulsifiers, detergents, adhesives, and enhanced
oi1 recovery fluids.
A microbial ~iopolymer is generally obtained by either
fermentative or enzymatic synthesis. In fermentation, a
microorganism that is ~enetically capable of building a
specific polymer is placed in a medium with the necessary
nutrients and substrate. The polysaccharide is recovered fro~
this medium. In enzymatic $ynthesis, microbial cells are
withdrawn from a cell culture, leavin~ a culture fluid
containing the extracellular enzyme. The enzyme is then
contacted with the substrate to produce the polysaccharide.
Further information on polysaccharides and their manufacture
can be found in "Microbial Polysaccharides," Kirk-Othmer
Encyclopedia of Chemical Technolo~, 3rd Ed., Vol. 15 at pages
439-5~.
Synthetic polymers are prepared by the combination of
one or more types of monomeric materials under conditions
~ .
suitable to polymerization. They are generally less complex
than biopolymers, usually consisting of an orderly progression
of simple monomer units.
80th biopolymers and synthetic polymers can be added
~ to water or other solvents in order to form solutions having
; beneficial characteristics, such as increased solution
viscosity. The viscosities of these solutions can vary ~reatly
depending the amount of the polymer in solution, the molecular
weight and conformation of the polymer, temperature, the
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presence of salts (especially multivalent salts such as
magnesium and calcium), and other factors.
The continued stability of polymer solutions under
conditions of severe temperature, salinities, pressures, shear
forces, and the presence of oxygen and chemicals is a major
concern for some applications. Some microorganisms, whether
naturally present in the solution environmen~ or introduced by
contamination, are also capable of degrading polymeric
substances and thereby decreasing the solution viscosity. For
example, many microorganisms secrete enzymes (cellulase,
amylase) that can hydrolyze polysaccharides into
monosaccharides that can be used in the organism's metabolism.
Synth~tic polymers can also lose viscosity under microbial
attack.
For the reasons given above, one of the objects of
current research i5 to produce stable polymer solutions. The
solution must be stable to the chemical, thermal, and
biological conditions encountered in various industrial
processes. Of parti ular interest is the search for a polymer
solution for use in enhanced oil recovery that will maintain
its stability during pretreatment steps, injection, and
throughout the reservoir,
For economic reasons, another object is to obtain the
maximum viscosity for the least cost, i.e., the most viscosity
from the least amount of polymer in the solution.
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SUMMARY OF THE INVENTION
The invention concerns a method for enhancing the
viscosity of a solution of preformed polymers by combining that
solution with a microorganism ineapable of de novo synthesis of
said polymers but capable of increasing the viscosi~y of said
polymer solution, and maintaining that solution at conditions
favorable for microbial growth. The viscosity of this solution
is increased or stabilized relative to a solution which does
not contain the microorganism.
The microorganisms used to increase the viscosity of a
chosen preformed polymer can be selected by using a microbial
screening process characterized by
(a) preparing a growing culture of the microorganism;
(b) preparing an aqueous solution of the preformed
. polymer and adding nutrient~ and minerals
required for microbial growth;
~c) separating that solution into two portions: a
test and a control;
d) inoculating the test solution with the culture oE
: the microorganism and incubating the mixture; and
(e) measuring~the viscosities of both test and
. : ,
, : control solution~ after incubation to determine
if the test solutlon is relatively more viscous
than the control.
DETAILED DESCRIPTION
It has now been discovered that certain microorganisms
are capable of increasing ~he viscosi~y of a polymer solution
:
~ : even when that microorganism is itself genetically incapable of
: ~ :
de novo synthesis of the polymer. The term "de novo" is used
in this patent to exclude those known techniques where a
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nicroorganism contructs polymeric material (such as polysac-
charides) from monomers, and thereby increases the viscosity of
a solution. As this invention is defined, the microorganism
cannot build the chosen polymer from ~ono~ers. This method is
therefore most appropriate for preformed polymers (i.e.,
polymers which have previously been produced by either chemical
or microbial syntheses). For example, a particular micro-
or~anism can be ~enetically incapable of manufacturing a
polysaccharide, but is capable o~ alteringithat a solution of
that polysaccharide to effectuate an increase in its viscosity.
While not`intending to be bound by a particular
theory, it is believed that the microor~anism acts to increase
the avera~e molecular weight oE the polymers in solution,
possibly throu~h ehe action of one,or more enzymes. A detailed
description of suitable polymers, microorganisms, viscosity
chan~es, and the process is given below.
Polvmers
_
The poly~ers useful in this invention include any
biopolymers and synthetic polymers that are compatible with
water. Preferred polymers are not only compatible with water,
but are readily solu~le in water because they will thus be
easily accessible to the microor~anism.s and any accompanying
. .
extracellular enzymes in an aqueous media. However, even
partially-soluble or water-insoluble polymers are-contemplated
~ to be within the scope o~ this invention, provided t~at an
; interfaee exists between the polymer and the microorganism or
~` enzymes.~
In the preferred practice of the invention, the
polymer can be chosen from those known to be effective for a
particular end use. For example, a polyacrylamide can be
,
* For the purposes of t~i's invention, a "solut~on" of polymer
means, in a~dition to dissolved polymer, any ~ater weta6le polymer
surface such as polymer suspensions, colloidal dispersions, polymeric
gels or emulsions, and weta~le polymer;~c solids.
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chosen if the desired use is water treatment, enhanced oil
recovery, mineral processing, or pulp and paper processing.
Extensive information has been published on
applications for polymer solutions. Lon~-chain water-soluble
polymeric materials are extensively used as flocculation aids
for solid-liquid separations. The use of aminated starches,
polyamines, and polyacrylamides in industrial water treatment
is summarized in "Water (Industrial)", Kirk-Othmer Encylopedia
of Chemical Technolo~, 2d Ed., Vol.22. Other polymer
flocculants are used in processes such as water clarification,
sewage treatment, metal finishing, paper production, sugar
refinin~, mineral extraction, and food processing as discussed
in "Flocculating Agents", Kirk-Othmer Encylopedia of Chemical
TechnoloRy, 3d Ed., Vol.10.
` .Enhanced oil recovery (EOR) usually requires the
injection of an aqueou~ fluid, s~ch as water or brine, to push
~;the oil ahead of it through the formation to a production
well. A polymer is often added to at least a portion of the
injected Çluid in order to form a slug having increased
viscosity. The polymer-thickened fluid moves in an even front
and minimizes the by-passing and f in~ering that mi~ht otherwise
occur during conventional waterflooding.
; Polymers considered to be useful for enhanced oil
recovery (EOR) include guar ~um, hydroxypropyl guar, sodium
carboxymethyl cellulose,hydr~ethyl cellulose, carboxymethyl
.~
hydroxyethyl cellulose, xanthan gum, glucan, locust bean
~u~, ~olyacrylamide, hydrolyzed polyacrylamide, poly(acrylic
:: ~
~ acid) and salts~poly(2~acrylam~do-2-methyl propone sulfuric acid)(2
; salt) ~AMPS), poly(acrylic acid-co-acrylate ester), poly (vinyl
: :: pyrrolidone), cellulose sulfate esters, poly (ethylene oxide),
poly (vinyl alcoholj, polyamine, poly(vinyl acetate-co-maleic
anhydride3, and poly (styrene sulfonic acid) and salts.
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Preferred among these are hydroxyet~yl cellulose, carboxymethyl
hydroxyethyl cellulose, xanthan gum, and hydrolyzed
polyacrylamide.
The concentration of the polymer solution used in EOR
is generally determined by the permeability of the rock and the
viscosity of the oil in the reservoir. For economic reasons,
the concentration is kept to the minimal effective levels,
which are typically between 20 ppm and 3000 ppm, more typically
50 to 1000 ppm and most typically 200 to 600 ppm. High
temperatures, shear stresses, high salinity, extreme pH values
are other reservoir conditions which must be considered in
choosing an ef~ective polymer.
Polymers are otherwise used in oil fields in drilling,
cementing, fracturing, acidizing, controlling water production,
. preventing sand production, clay stabili~ation, lost
circulationf and the like. Guar, guar derivatives,
; cellulosics, xanthans, locust bean gums, starches, and
; synthetic polymers such as polyacrylamide are commonly used, as
more fully described in Chatterji, J. and Borchardt, J.K.,
"Applicstions of Water-Soluble Polymers in the Oil Field", J.
: Pet. Tech. 2042-2056, (Nov. 1981).
Preferred polymers for EOR mobility control agents
~:~ will have all or most of the following character;stics :
1~ Ready solubility in water with a high positive
:~ : affect on solution viscosity;
: 2. Tolerance of dissolved salts (brine) in the
,
aqueous solvent;
3. High molecular weight for maximum viscosity
effect;
4. Devoid of excessive brsnching or gels that might
cause injection problems;
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S. Stability under thermal, biological, and shear
s~resses in the reservoir environment and
injection process;
6. Potentially inexpensive in large volume
production or having a high volume natural source.
M_asurement_of Viscosit~
The viscosity of a water-soluble polymer in solution
is a function of many factors, chiefly molecular weight and
electrolytic character. The greater the molecular weight of
the polymer, the greater is the viscosity. Char~ed polymers
also tend to lose viscosity in highly saline water due to
neutralization of the charges by the ions in solution.
The classic definition of viscosity for Newtonian
fluids will be employed in this patent, where viscosity equals
shear stress over the rate of shear. Shear stress is further
defined as the force per unit area required to produce the
~; shearing act;on. The rate of shear is a measured value.
A suitable way to measure viscosity is to employ a
;~vi~scometer such as one manufactured by Brookfield Engineering
Laboratories, Inc., of Stoughton, Massachusetts. The
~, ~roo~field Viscometer measures viscosity by measuring the force; required to rotate a spindle in a fluid. Because practically
all fluids become thinner~ as temperature increases and thicker
as they cool, the temperature of the fluids being compared
should~ be recorded and~kept the same if possible.
; ~ The Broo~field LVT Viscometer with the UL (Ultra-Low
viscosity~ adapter is especially suitable for this
application. The UL a~dapter provides amplifyin~ effects which
.- : : :
makes poss~ible measurements ~ith a reproducibility of
0.2;centipoises (cps)~ in the ultra-low viscosity ran~e of
~; û to 10 cps. Calibration of the instrument showed an average
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error of +0.2 cps at both ends of the shear rate range (10
sec~l and 70 sec~l). The shear rate on the instrument can
be varied from 73.42 to 0.36 sec 1, which covers the range of
shear encountered underground in the flooding process
~typically 10 sec 1),
Other measuring devices such as capillary viscometers,
plate and cone viscometers, and concentric cylinders can be
used as appropriate.
- Some polymer solutions will exhibit thixotropic
properties, characterized by the ability of a solution to
become fluid when subjected to shaking, stirring, or other
stress and then return to a gel when allowed to stand. Common
examples of this are mayonnaise and paint. Thixotropy is also
desirable for polymer solutions used in enhanced oil recovery,
in which the solution is more fluid to facilitate pumping but
becomeæ more viscous once it enters the oil-bearing formation.
-~ Non-Newtonian fluids are those in which a change in
the rate of shear is not proportional to a change in the shear
stress. For such fluids, the relationship between sheer rate
and shear stress is sometimes determined at several rates of
shear. Although the polymeric solutions in this invention may
~:
~;~ be non-Newtonian, shear rates which are obtained using a single
; ; set of conditions for shear stress will usually be sufficient
to use as a basis of comparison to determine any change in
viscosity. Additional measurements of shear rate with
d~ifferent shear forces can be made if desired. Thus for these
- ~ purposes, all comparisons ar`e made as if the fluids being
measured behaved as Newtonian fluids. Procedures for
~ , :
~ determining viscosity are easily within the skill of the
- analytical chemist, and more detailed guidelines can be found
in textbooks, journals, and viscometer manufacturers'
instructions.
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For ~he purposes of this invention, a change in
measured viscosity of about 10 percent is considered to be very
- signi~icant when comparing a test polymer solution to a control
solution. Since commonly-available equipment is incapable of
accurately measuring the weight of polymers with a molecular
weight approaching one million, the change in the polymer was
measured indirectly by determining intrinsic viscosity.
The mo~t common method of estimating the molecular
weight of a polymer in solution is the use of "intrinsic
viscosity" lN]. The intrinsic viscosity "[N]" can be related
to the molecular weight of the polymer by means of the Mark,
Houwink~ and Sakurada equation:
N] = KMa
where K and a are constants being a function of the polymer
type, temperature, and solvent. (This equation is discussed in
more detail in Billmeyer (see belowl.
~';
~, . -
The constants are initially determined by measuring
polymers of known molecular weight. The determination of [N]
is-made by capillary viscometry of dilute polymer solutions at
several concentrations. Viscosity data is extrapolated to zero
concentration in order to produce a value related to molecular
weight at a constant polymer ~ype, solvent, and temperature.
Even if constants are not known and the absolute
: ~alues of molecular weight are unknown, the procedure is still
- useful because the vaiue o~ [N] is directly proport;onal to a
low power of thc molecular weight. For a solvent-temperature-
polymer system that does not have the constants available, the
values of [N] can be used to determine increases or decreases
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of molecular weight, but not the actual value~. This concept
is discussed further in a popular text (Billmeyer, Jr., F.~,
Textbook of Polymer Science, 3rd Ed., New York: Interscience
Publishers. 1965; pp. 79-85). Since only relative changes in
molecular weight need to be determined, this procedure is
satisfactory.
icroorganisms
The microorganisms useful in this invention will have
the capability of increasing or at least stabili7ing the
viscosity of preformed polymers in solution.
In the broadest aspect of the invention, a suitable
microorganism can be chosen by selecting one or more candidates
and performing the screening test described below for
determin;ng the effect of the microorganism on the polymer
solution. Candidate microorganisms can be found almost
anywhere. Soil samples and plants are logical locations, ~s
well as subterranean and surface water, Oxygen availability
should not be a limiting factor in the selection, since both
aerobes and anaerobes are useful. Existing cultures of
microorganisms can, of course, be tested for utility in this
invention.
~ lasses of microorganisms contemplated as useful in
this invention include phototrophic bacteria, gliding bacteria,
sheathed bacteria, budding or appendaged bacteria,
spiral and curved bacteria, gram-negative aerobic rods and
cocci, gram-negative facultatively anaerobic rods,
gram-negative anaerobic bacteria, gram-negative cocci and
coccobaccilli, gram-negative anaerobic cocc;, gram-negative
chemolithotrophic bacteria, methane-producing bacteria,
gram-positive cocci, endospore-forming rods and cocci,
gram-posi~ive, asporogenous rod-shaped bacteria, and
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actinomycetes and related organismsO Those classes unlikely to
be useful in this invention include rickettsias and
mycoplasmas. Further information on the characteristics of
each of these classe~ can be found in Be~gyls Manual f
Determinative Bacteriolo~y ~8th Ed.) R.E. Buchanan and N.E.
Gibbons, Co-editors (1974).
Once the sample has been obtained, the microorganism
should be isolated and propagated on one or more types of
laboratory media using standflrd techniques.
Process
The invention may be practiced using any production
methods suitable for microbiological synthesis. Conventional
methods used in the art are divided into two categories: batch
and continuous. In a batch process all of the starting
materials including the microorganisms are placed in a vessel
where they remain until the desired product is formed,
whereupon the vessel is emptied and the product is separated.
In a continuous process the raw materials are added and the
product is withdrawn at a steady rate. A continuous method is
preferred for making or treating large amounts of material,
although batch processes are especially useful when close
control over the process conditions is desired.
The process is typically operated at condi~ions which
are optimal for ~rowth of the microorganism, since the maximum
results are obtained at a hi~h metabolic rate. These op~imal
. conditions are usually similar to those found in the
microorganism's natural environment. The environment is
duplicated i~ possible to provide the same temperature, pH,
salinity~ trace elements, and other materials.
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If the environment cannot be readily duplicated, a
standard nutrient media should be used to provide water,
sugars, amino acids, vitamins, minerals, and trace elements.
Such media are available at biological supply houses.
The optimal range of pH and temperature for rapid
growth~ as well as the range of tolerable conditions, can
easily be determined by common tests known to those in the
art. A more detailed description of these tests is given in
"Growth", Ralph N. Costilow, Ed. Manual of Methods for General
Bacteriolo~y pp. 65-179, (1981).
Screening Procedure for Microbial Viscosity Enhancement
The following procedure can be used to screen
candidate polymers to determine if a given microorganism will
enhance the viscosity o a solution of that polymer. It can
also be used to screen candidate microorganisms to determine if
they wiIl enhance the viscosity of a given polymer, The
procedure can also be used to screen entirely new combinations
of polymers and microorganisms.
A. _reparation of Culture. A growing culture of the
microorganism is prepared in a suitable growth medi~m. The
specific pH and ingredients of the medium will vary depending
upon the requirements of the particular microorganism. This
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culture is preferably incubated for twenty-four hours at the
organismls optimum growth temperature.
B Inoculation of the Polymer Solution. The polymer solution
i5 preEerably concentrated enough to provide measurable
viscosities for the viscosity test, but sufficiently dilute so
that the amount of polymer is not toxic to the microorganism.
The polymer solution can be sterilized by steam or other means
to avoid the effects of competing growth of undesired microbial
contaminants. A basal salts solution is optionally added to
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the polymer to assure that essential nutrients and mineral~ for
the microorganismO
The growing cell culture can then be centrifuged,
washed to remove any residual growth medium, and resuspended in
a minimal amount of distilled water. For optimal results, a
cell count is performed to determine the concentration of
cells. A measured amount of inoculum, containing a given
concentration oE cells, is then added to the solution
containing the preformed polymer. A control solution is al80
prepared which has the same concentration of polymer solution
and basal salts, but does not contain any microorganisms. An
equivalent volume of sterile distilled water i~ added to the
control to match the volume of inoculum added to the solutions
containing the prefermed polymer.
C. Incubation of_the Solution. The control and the polymer
solution with the microorganism are incubated at the optimum
growth temperature for the microorganism. A sample of the
solution can be removed periodically to show trends in any
viscosity change, if desired. Definitive results are typically
Obeained after ao incubation of 2 to 7 days.
After incubation, the microorganisms are preferably
remoYed by centrifugation (or other methods compatible with the
.
polymer and microorganism), leaving a microorganism-free
~solution~of the polymer.
D. Determination of Chan~es in Viscosity.
The viscosity of the microorganism-free solution can
.
~ ~ be determined by the methods discussed above, such as a
,
Brookfield visco=eter with an ultra-low viscosity cell which
will produce viscosi~y data (centipoise) at a variety of shear
rates. The ViSCOsity of the test solution is compared to the
viscosity of the con~rol solution. If the viscosity of the
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test solution is greater than that of the control solution, the
microorganism can be considered capable of increasing the
viscosity of a solution of that polymer.
SPECIFIC EMBODIMENTS
Several microorganisms were screened to determine
their effect on polymer solutions, using the following general
procedure except where specifically noted.
'
Preparation of a Medium. A basal salts medium was prepared at
a double-strength concentration for later dilution with the
appropriate polymer solution. The basal salts solution
consisted of the following:
~ Yeast extract O.l g
- ~ K~U PO4 1.0 g
KH2 P4 1.0 g
(NH4) 2S4 ~ ~ o
Ca~C12 2H20 Oo l g
Deionized H20 500 ml
,: :
The exact composition of the basai salts medium is
usually not critical9 and~it can be altered depending upon the
equirements~of the microorganism~being tested. The same basal
salts medium shoùld be used throughout a similar series of
viscosity tests9 however,~because the presence Oe salts can
affect the viscosity characte;ristics of a polymer in solution.
The CaC12 2H20 was autoclaved separately to
avoid pre~cipitation with the other components upon heating.
After both solutions had cooled to room temperature, the
CaC12 2H20 was added to the other ingredients. The
~ ba~sal salts medium was checked for purity by streaking an -
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aliquot onto a nutrient agar plate and incubating the plate at
3~C Eor 24 hours.
Preparation of a Culture. Bacteria and fungi to be screened
were grown on nutrient agar slants and Sabouraud dextrose agar
slants, respectively. The microorganisms were incubated at
30C for 24 hours, aerobically and/or anaerobically depending
on their physiological properties. Culture purity was
determined via the gram stain and microscopic examination.
Five ml of the basal salts medium were added to each slant and
the organisms were removed from the surface by vortexing. Five
hundredths of a milliliter of the cell suspension was used as
the inoculum.
'` '
Pre~ration of the Polymer Solution. A quantity of the polymer
was added to deionized water to achieve a concentration of 2400
ppm (weight to volume). The polymer solution was adjusted to
pH 7.0 and was s~team sterilized at 15 psi nd 121C for 60
minutes. The solution was then tested for purity as described
for the basal salts solution.
Incubation.~ The basal salts solution and polymer solution were
mixed in a 1:1 ratio by volume to provide a tes~ solution of
five ml. Five hundredths of a milliliter of microbial
.
suspension (inoculum) was added to each of ~hree tubes. The
tubes were incubated for 14 days under conditions described for
the inoculum preparation. After incubation each tube was
examined for turbidity indicative of microbial growth.
IntrInsic Viscosity. For these experiments, results were
used for comparative changes of molecular weights. Low
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molecular weight metabolic products were not removed and were
assumed to make no contribution to viscosity. Because of the
washing procedure to remove the microbial cells from the
polymer solution, any viscosity change can be a~tributed to an
alteration of the polymer itself and not to the presence of
biological residue. The temperature for the intrinsic
viscosity determination was 25C.
EXAMPLES 1-4
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A gram negative rod characterized as a strain from the
genus ~ was isolated from oil field produced water
and was assigned code number M06882. In earlier lab
experiments this strain had demonstrated the ability to use the
partially hydrolyzed polyacrylamide, Dow Pusher(R) 500, as
its sole carbon source for growth.
The media used in this experiment were prepared in two
parts. A first polymer solution was made by dissolving Dow
Pusher(R) 500, as obtained commercially in distilled water at
a concentration of 2400 ppm (weight to volume) and pH 7Ø The
second solution was made in the same way, but af~er purifying
the polymer by alcohol extraction. Both solutions were stirred
for 24 hours, weighed before sterilization, and then autoclaved
(sterilized) for 1 hour at 121C and 15 psi. All water lost
due to autoclaving was returned by adding sterile distllled
water~until the polymer solutions reached their original weight.
A basal salts solution was prepared at a double
concentration, as explained above. It contained 0.02% yeast
extract manufactured by BBL (a division of 8ecton, Dickinson &
Company of Cokeysville, MD), 0.2% KH2P04, 0.2% K2HP04,
0.1% (NH4)2 S04, and 0. ~ CaC122H20 in distilled
water at pH 7Ø The CaC12 was made as a separate stock
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æolution and added to the salts solution after both were
sterilized and cooled to room temperature.
The two separate Dow Pusher(R) 500/basal salts
solutions for this experiment were made by aseptically mixing
1:1 solutions of basal salts medium (2X) and 2400 ppm (w/v)
polymer solution.
The Enterobacter M06882 culture was grown aerobically
on a nutrient agar (Difco) slant at 30C for 24 hours. The
cells were harvested by adding 5 ml of sterile basal salts
solution to the test tube slant and vortexing the tube gently.
This cell suspension was used as the inoculum for this
experiment.
All bottles were incubated at 30C for fourteen
days. Aerobic incubation included rotary shaking of the flasks
at 200 rpm. Anaerobic incubation was carried out in an
anaerobic chamber system ~manufactured by Forma Scientific).
Control solutions were incubated both aerobically and
anaerobically.
After incubation, all test solutions and,controls were
centrifuged (lO,000 x g, 30C, 20 min) and the supernatant
aseptically decanted into clean, sterile sample bottles. The
viscosities of the solutions were measured.
The molecular weight of the Dow Pusher~R) 500 was
assumed to be 3.4 x 106, based upon published data (F.D.
Martin and J.S. Ward, Polymer ~ nts, Vol. 22, No. 2,
~merican Chemical Society ~eeting, August, 1981, p.24). The
control solution viscosity was also assumed to be constant.
~etailed data showing measured viscosi~y~ intrinsic viscosity,
ànd estimated molecular weight of the polymer for control
solutlon A and Examples 1 and 2 are in Table 1.
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Table 1
Enterobacter M06882 with Dow Pusher(R) 500
Intrinsic Measured
Viscosity Viscosi~y (cps) Estimated
Example Incubation [N]10 sec~l 70 sec~l Mol. Wt.
A. Aerobic 16.6 9.7 7.0 3.4 x 106
1. Aerobic 18.1 9.2 6.3 3.7 x 106
2. Anaerobic 27.6 17.1 7.3 5.7 x 106
Compared to the control, the organism causes an
- increase in viscosity or stabilizes the purified polymer from
-- decomposition over the 14 days. Under anaerobic conditions,
Enterobacter M06882 increases the apparent molecular weight of
Dow Pusher(R) 500 from 3.4 x 106 to 5.7 x 106, an
increase of about 68%. Estimates of molecular weight were made
from changes in intrinsic viscosity.
~, ~
EXAMPLES_5-8
- ~ The procedures above were repeated to determine
changes in intrinsic viscosity as dlscussed above. All
conditions were the same except where noted.
- Table 2 summarizes the intrinsic viscosity results of
these solutions incubated with Enterobacter M06882 for 2
veeks. Relative viscosity increases were found in all samples
tested. The greatest~enhancement in solution viscosity was
; with the samples that were incubated anaerobically. Because a
control was not available to compare the effec~s of a purified
polymer under anaerobic incubation, the effects of puriEication
were deemed secondary. Examples 2 and 6 were therefore
compared to Control C, the closest comparison.
The intrinsic viscosity data from Examples 1-4 and
Controls A and B are also shown in Table 2.
:
19 .
7~5~ ~
(5792)
Table 2
Enterobacter M06882_and Polyacr~l~m de*
r ' Incubation Intrinsic ~0 Increase
Exa~le_ _Polymer Condi~ions __ Vis~ yL_____Viscosit~
A. Pure Aerobic 16.6 CONTROL
B. Comm. Aerobic 15.5 CONTROL
C. Comm. Anaerobic 16.9 CONTROL
_________ ______________________________________________
1. Pure Aerobic 18.1 9
2. Pure Anaerobic 27.6 63
3. 50mm. Aerobic 17.0 10
4. Comm. Anaerobic 26.6 57
5. Pure Aerobic 18.1 9
6. Pure Anaerobic ~17.8 5
7. Comm. Aerobic 18.5 16
8. Comm. Anaerobic 19.1 18
*Dow PushertR) 500: 'IComm.'' means as commercially obtained;
Pure" means the polymer was purified by extraction
~; with methanol.
:~
:
~: ~ :: :
: ::
:
~ .
.~
~ 20.
5~ ~
(5792)
EXAMPLES 9-130
. . .
Eight st~ains of bacteria in addition to Enterobacter
M06882 were chosen for testing. All were capable of growing
aerobically and/or anaerobically on various water-soluble
polymers. The nine strains are identified by the following
reference numbers:
Gram negatIve rods: M06882
P040~2
P03482
P04282
- 20582
Gram po~itive cocci: M06682
M06782
M06182,
` Gram positive rod: 16782
The organisms P04082, P03482, and P04282 were laboratory
contaminants isolated from aqueous polymer solutions. All
other bacteria were isolated from oilfield produced water and
;~ drilling muds.
All strains (except M06182) were grown aerobically in
~; nutrient broth (Difco) at 30C for 24 hours. Organism M06182
was incubated anaerobically at 30C for 24 hours using
thioglycollate broth.
After~incubation, the bacteria were washed three times
by centrifugation (5,000 x g, 5C, 10 min) with sterile
distilled water. The washed cells were resuspended in minimal
sterile distilled water. Cell counts were done on each cell
` suspension using a Petrof-Hausser counting chamber.
Medis were prepared for this experiment in two parts.
Polymer solutions were made in dis~illed water at a
concentration of 2400 ppm tw/v) and pH 7Ø The polymers in
Table 3 were used in their commercial form and after
purification by extration.
; 21.
.~
7 ~50
(5792)
Table 3
Polymer Source
Polyacrylamide, hydrolyzed Dow Pusher(~
Polyvinyl alcohol Polysciences, Inc., Cat. #2815
Poly~AMPS] poly(2 acrylamido-2-methyl
propanesulfonic acid) from
Lubrizol Corporation
Xanthan gum Xanflood(R); Kelco
Hydroxyethylcellulose Natrosol 250 HHR, from
Hercules, Inc.
Carboxymethylcellulose CMC 9H4 from Hercules, Inc.
Glucan Actigum CS 11-L obtained
from Jetco Chemicals, Inc.
Polyacrylamide synthesized unhydrolyzed
polyacrylamide; moIecular
weight of 3.5 x 10
Poly(acrylic acid) Scientific Polymer
Products, Inc., catalog
All solutions were stirred for 24 hours, weighed, and
then autoclaved (sterilized) for 1 hour at 121C and 15 psi.
All water lost due to autoclaving was returned by adding
sterile distilled water~until the polymer solutions reached
their original weight.
A basal salts medium was prepared as described in
Example~l. Eighteen differene~polymer media for~this
exper~iment were ma~de by aseptica~lly mixing 1:1 (v/v) solutions
-~ ~
of basal salts medium and 2400 ppm polymer solution.
Fifty ml~aliquots of~polymer media were aseptically
tran~sferred to clean, sterile 150 ml Erlenmeyer flasks. Each
; Elask~(except controls) was: inoculated to a concentration of
103 bacteria/ml with the appropriate cell suspenslon.
Control~s were inoculated wi~th equivalent volumes of sterile
:~
~ distilled water.
.:
~: :
, ~
22.
.
7~5~ ~
(5792)
All flasks were incubated at 30C for two weeks.
Aerobic incubation included rotary shaking of the flasks at 200
rpm, and anaerobic incubation was conduc~ed in an anaerobic
chamber system. Controls ~ere incubated aerobically.
After incubation, all test solutions and controls were
centrifuged (10,000 x g, 30C, 20 min) and the supernatant
aseptically decanted into a clean, sterile sample bottle.
Apparent viscosity determinations were made using a Brooksfield
viscometer. Table 4 s~ows the results for Examples 9 to 130,
and also incorporates data from the Examples 1 to 8.
: '
TABLE 4
Selected Microbe/Polymer Combinations
Percent
Incu- Change in
ExamplePolymer Or~anism bationl Viscosity2
1Polyacrylamide3, puri~ied MO6882 A 9.
2 " " MO6882 ~n 63.
;~ 3Polyacrylamide3 (comm.) MO6882 A 10.
4 " " MO6882 An 57.
5PolyacryIamide3, purified MO6882 A 9.
6 " " MO6882 A 5.0
"
7Polyacryl;amide' (comm.) MO6882 A 16.
8 " MO6882 An 18,0
9 " P04082 A -8.0
:,~
" P04082 An +3.5
1l " P04282 A -4.4
~ ~ I2 " P04282 An +4.4
; 13 ll 16782 A
14 " ~ 1~782 An -28.3
" 20582 A -~6.2
,:
23.
~ '71~LS~3 ~
(5792)
TABLE 4 (continued)
Selected Microbe/Po~,ymer Combinations
Percent
Exam~le . Polymer Organism bation Viscosity2
16 Polyacrylamide3 (comm.) 20582 An ~4.4
17 " (pu~ified)4 P04282 A -1.0
18 " P04282 An +35.6
19 "
P04082 A +4.8
" P04082 An +9.6
21 Polyvinyl Alcohol M06682 An -7.7
22 " MO6682 A 0
23 " M06882 A 0
24 " MO6882 : An 0
" P04082 A 0
26 " PO4082 An +7.7
27 " 16782 A 0
28 " 16782 An -7.7
29 " 20582 A -7.7
.,
~ : 3~n ~ : 20582 An -7.7
3I " ~ P04282 A +7.7
: 32 " ~ P04282 An +7.7
33~ Poly~vinyl Alcohol~(purified) M06682 A 0
:: 34 " : ~ ~M06882 A -7.7
" ~ MQ6882 An 0
36 " P04082 A 0
37 ~ Po4082 An 7.7
38 : " ~ P034B2 A 0
39 " ; PO3482 An -7.7
~ " P04282 A 0
:41 " PO4282 An +7.7
~ :
'~: :
:~ 24.
7~ 15~ ~
(5792)
TABLX 4 (continued)
Selected Microbe/Polymer Combinations
Percen t
Example ~ Or~an i sm ba-t i onVi sc~2
42 Poly (AMPS) M06882 An -3.8
43 " M06882 A +0.6
44 " P04082 A -8.3
" P04082 An +1.9
46 " P04282 An 0
47 " M06682 A +7.0
48 Poly(AMPS), purified6 M06682 A -1.9
49 " M06682 An -0.5
" M06882 A -12.9
51 " M06882 An -10.0
52 " P04082 A -29.7
53~ ;" P04082 An -29.2
54 ~" P03482 A -25.4
!I P03482 An -23.4
56 " ~ P04282 A -27.3
57~ " P04282 An -19.6
.
58~ Xant~an Gum P04082 An -3.0
59 ~ " ~ P04282 A -3,0
;60 " ~ ~ P.04282 An -6.1
61 ~ 20582 An -3.0
62 ~Xanth~an ~um, purified4 P04082 An 0
; 63 " ~ P04082 A +6.7
64 " 16782 An 0
~ 65 i' ~ ~16782 A ~6.7
: .
~ 25.
7~ ~5~
(5792)
TABLE 4 (continued)
Sele ~
Percent
Example Polymer Or~anism bation Viscosit~2
66 Hydroxyethylcellulose M06882 A -10.7
67 " MO6882 An -10.7
68 " . P04082 A 0
69 " PO4082 An -7.1
" P04282 An -4.3
71 " (purified)4 M06882 A -10.0
72 " MO6882 An -13.3
73 " P04082 A -16.7
74 PO4082 An -3.3
" P04282 An -6.7
76 Carboxymet~ylcellulose M06882 A -8.3
77: " P04082 A -11.1
78 " : ~ P04282 An -11.1
79~ "~(purified)4~ M06782 A -3.3
~ MO6782 An -10.0
81 " M06882 A -3.3
8~ P04082 A -3.3
83 " ~ ~ PO4082 An O
84 :~ P04282 An -6.7
8:~ : Glucan M06882 A -10.5
86 : :: MO6882 An -6.7
;87 ~ : P04082 A -7.7
88 ~ PO4082 An -3.4
89 " ~ P04282 A +15.9
:90 ~ " ~ 16782 A +17.4
91 " : 20582 A ~16.1
26.
7~
(5792)
TABLE 4 (continued)
Selected Microbe!Polymer Combinations
Percen t
ExamE~le Poly~ Or~anism bationViScosit~2
92 Glucan P04282 An -17.2
93 " 16782 An -18.1
94 " 20582 An -19.8
" (purified)4 M06882 A -18.2
96 " M06882 An~34.7
97 " P04082 A -8.5
g8 " P04082 An+51.4
nn "
77 P04282 A ~19.4
100 " P04282 An -13.8
101 ~ " P03482 A ~33.4
: io~ ~ 16782 An -13.0
103 " 16782 A +46.6
104 " 20582 An -13.6
105 " 20582 A +16.0
106 Polyacrylamide5 M06882 An -5.3
, :
107 " P04802 A ~5.8
108 " ~ P~4802 An 0
109 " ; P04282 A -10.5
110 " PO4282 An -5.3
111 " M06182 An -10.5
112 Polyacrylamide (purified)4 M06882A -13.6
~: ::
113~ " P04082 A -~7.1
1 114 " PO4082 An -4.5
115 " P03482 A -18.2
~: :
116 " PO3482 An -4.5
117 " P04282 A -4.5
: 118 ll PO4282 An -9.1
27.
~ ~ ,
~ ~L~7~
(5792)
TABLE 4 (continued)
Selected Microbe ~ mbinations
Percent
Ex~ple Polymer Or~anism b~tionVi~c~ 2
119 Polyacrylic Acid7 M06882 A -3.3
120 " M06882 An +3.3
1~1 " P04082 A -3.3
122 " PO4082 An -3.3
123 " P03482 An -3.3
124 " P04282 An ~16.7
125 " (purified) M06882 A -3.3
126 " MO6882 An -6. 7
; 127 " P040g2 A -3.3
128 " PO4082 An -6.7
~- 129 " P04282 An -3.3
~130 " PO4282 A +16.7
1 "A" denotes Aerobic; "An" denotes anaerobic
2 Intrinsic viscosity; indicates relative change in molecular
we1gh~ of the polymer compared to the control
3 Dow Pusher(R) 500, a partially-hydrolyzed a~rylamide.
;~ 4 Purified by~extraction with methanol
., r
' Synthesized in;~Sohio~laboratories; unhydrolyzed polyacrylamide
with 3 molecular weight of 3~.5 x 105.
6 ~ Purified by ~extraction~ with isopropanol.
7 Purified by ext~raction with acetonitrile.
, .
~ 2~ .
.
lS~) ~
(5792)
Microbial Enhancement o~ Polymer Solution Viscosity
Several samples tested in this polymer/bacteria screen showed
solution viscosity increases after 2 weeks of incubation. Some of
the samples that showed microbial enhancement of polymer solution
viscosity can be found in Table 5, grouped according to the code
number for the bacterlum.
;` ' -
' ~ ~
'
::
` '
29 .
,
7~5~ ~
(5792
TABLE_5
Percent
Example ~ Or~anism bationViSco~it 2
1 Polyacrylamide3, purified MO6882 A ~9.
" MO6882 An ~63.
3 Polyacrylamide3 (comm.) MO6882 A ~190
7 ,, ,' MO6882 An ~16
8 " MO6882 An tl8.o
43 Poly(AMPS) MO6882 A ~0 6
96 Glucan (purified)4 M0S882An +34 7
120 Polyacrylic Acid MO6882 An +3.3
Xanthan Gum, purified4 16782 A +6.7
~ 90 Glucan 16782 A ~17 4
- 103 Glucan (purified)4 16782 A +46 6
47 Poly(AMPS) M06682 A +7.0
~- 15 Polyacrylamide, (Dow)3 20582 A +6~2
16 ' 20582 on +4.4
105 '' 255882_ A +16.1
101 : Glucan (purified)4 P03482 A +33.4
1:0 Polyacrylamide3 P04082 An +3 5
19 Polyacrylamide (purified)3 P04082A +4 8
:20 " " P04082 An ~9~6
26 Polyvinyl Alcohol PO4082 An +7 7
: 45 Poly~AMPS) PO408:2 An +1 9
~ 63: Xanthan Gum, purified4 po4082 A +6 7
-~ 98 Glucan (purified)4 : P04082 An+51 4
~ 12 Po7yacrylamide3 P04282 An ~4.4
;. 18 Polyacrylamide (purified)3 P04282An +35.6
31 Polyvinyl Alcohol P04282- A +7 7
32 "~ P04282 An ~7 7
41 " (purified):7 PO4282 An +7.7
89 Glucan :: ~ P04282 A +15.9
99~ Glucan tpurified)4 P04282 A +19,4
124 Polyacrylir Acid : P04282 An +16.7
130 ~ " (purified) : PO4282 A +16.7
" denotes~A ~ An"~denotes an~erobic
Intrinsic viscosity; indicates percent change in molecular
we~ight of the polymer relative to the control
: 3 Dow Pusher~R)~ 500.
4 Purified by extraction with methanol
7 Purified by:extraction with ace~onitrile.
--------------_______~_________ ___
~:
3~.
.
71L~5() ~
(5792)
Prior to this ~pplication or patent, the
microo~gan;sms in Table 6 were deposited with the Americ~n Type
Culture Collection (ATCC) in Rockville, Maryland, under an
agreement whereby they would become accessible to the public
upon ~he issuance o letters patent. A unique number is
~ssigned to ~ach microorganism culture deposited with the ATCC.
Table 6
Code Number vs. ATCC Number
: ~ MO6882 39553 _
PO4082 39555
PO3482 39560
PO4282 39558
2û582 39554
MO6682 39557
16782 39556
`; MO6182 _39559__
MO6782 39552
,
:~ :
'~
-``
;'
'
31.
