U.S. patent application number 14/374851 was filed with the patent office on 2015-02-05 for method of producing biosurfactants.
The applicant listed for this patent is GFS Corporation Aus Pty Ltd. Invention is credited to Michael Paul Bralkowski, Sarah Ashley Brooks, Stephen M. Hinton, David Matthew Wright, Shih-Hsin Yang.
Application Number | 20150037302 14/374851 |
Document ID | / |
Family ID | 48872831 |
Filed Date | 2015-02-05 |
United States Patent
Application |
20150037302 |
Kind Code |
A1 |
Bralkowski; Michael Paul ;
et al. |
February 5, 2015 |
METHOD OF PRODUCING BIOSURFACTANTS
Abstract
The present invention relates to a method of producing
biosurfactants, such as surfactin, comprising culturing at least
one biosurfactant-producing microbe in a liquid culture medium
comprising vinasse as a carbon source. Methods of using the crude
biosurfactant containing culture broth in tertiary oil recovery and
as antibacterial compositions in tertiary oil recovery are also
described. Methods of using the culture broth residue after
isolation of the biosurfactants as fertilizer and compositions for
this use are also described.
Inventors: |
Bralkowski; Michael Paul;
(Lexington, NC) ; Brooks; Sarah Ashley;
(Winston-Salem, NC) ; Hinton; Stephen M.; (Mount
Pleasant, SC) ; Wright; David Matthew; (Kernersville,
NC) ; Yang; Shih-Hsin; (Queensland, AU) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
GFS Corporation Aus Pty Ltd |
Naragba |
|
AU |
|
|
Family ID: |
48872831 |
Appl. No.: |
14/374851 |
Filed: |
January 25, 2013 |
PCT Filed: |
January 25, 2013 |
PCT NO: |
PCT/AU2013/000059 |
371 Date: |
July 25, 2014 |
Current U.S.
Class: |
424/93.46 ;
435/252.5; 435/71.3; 507/201; 530/321; 71/6 |
Current CPC
Class: |
A01N 63/10 20200101;
C07K 7/64 20130101; C12R 1/125 20130101; C05F 11/08 20130101; Y02A
40/20 20180101; C12P 21/02 20130101; C05F 5/006 20130101; C07K
14/32 20130101; A61P 17/00 20180101; C09K 8/584 20130101; A01N
37/18 20130101; Y02A 40/211 20180101; A01N 63/00 20130101; A61P
31/04 20180101 |
Class at
Publication: |
424/93.46 ;
435/71.3; 530/321; 435/252.5; 507/201; 71/6 |
International
Class: |
C07K 7/64 20060101
C07K007/64; A01N 37/18 20060101 A01N037/18; C05F 11/08 20060101
C05F011/08; C09K 8/584 20060101 C09K008/584 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 27, 2012 |
AU |
2012900312 |
Claims
1. A method of producing biosurfactant comprising culturing at
least one biosurfactant-producing microbe in a liquid culture
medium comprising vinasse as a carbon source, wherein the culturing
occurs at pH 6 to 8 and a temperature of 25.degree. C. to
40.degree. C.
2. A method of claim 1 wherein the at least one
biosurfactant-producing microbe is selected from the group
consisting of Bacillus subtilis, Bacillus licheniformis, Bacillus
amyloliquefaciens, Bacillus pumilus, Bacillus popilliae, RSA-203
and mixtures thereof.
3. A method of claim 2 wherein the at least one
biosurfactant-producing microbe is selected from the group
consisting of Bacillus subtilis, Bacillus licheniformis and
mixtures thereof.
4. A method of claim 2 wherein the Bacillus subtilis is Bacillus
subtilis NRRL B-3383, Bacillus subtilis RSA-203 or mixtures
thereof.
5. A method of claim 2 wherein the at least one
biosurfactant-producing microbe comprises a mixture of Bacillus
subtilis and Bacillus licheniformis.
6. A method of claim 1 wherein the vinasse is sugar cane
vinasse.
7. A method of claim 1 wherein the vinasse is present in the liquid
culture medium in an amount of from 3 to 10% w/v.
8. A method of claim 1 wherein the biosurfactant is selected from
the group consisting of surfactin, iturin, lichenysin, fengycin and
mixtures thereof.
9. A method of claim 8 wherein the biosurfactant is surfactin.
10. A method of claim 1 wherein the temperature is 30.degree. C. to
35.degree. C.
11. A method of claim 1 wherein the pH is 6.5 to 7.2.
12. A method according to claim 1 wherein the liquid culture medium
is inoculated with an inoculum of at least one
biosurfactant-producing microbe to provide an initial optical
density at 600.sub.nm of 1.2 to 1.4
13. A method of claim 1 wherein the liquid culture medium further
comprises a catabolizable nitrogen source.
14. A method of claim 13 wherein the catabolizable nitrogen source
is an ammonium salt.
15. A method of claim 14 wherein the ammonium salt is ammonium
nitrate.
16. A method of claim 1 wherein the liquid culture medium further
comprises at least one inorganic salt.
17. A method of claim 16 wherein the at least one inorganic salt is
a sulfate, chloride or phosphate of manganese, sodium or iron, or
mixtures of said salts.
18. A method of claim 1 wherein the culturing further comprises
aerating the liquid culture medium during culturing and collecting
foamate produced during aeration.
19. A method of claim 18 wherein aeration is begun before the
culturing is begun.
20. Biosurfactant produced by the method of claim 1.
21. A composition comprising at least one biosurfactant-producing
microbe and vinasse residue, wherein the vinasse residue is formed
by decomposition of vinasse by biosurfactant-producing microbes
during a culturing process.
22. A composition of claim 21 further comprising at least one
biosurfactant.
23. A composition of claim 22 wherein the biosurfactant is selected
from surfactin, lichenysin, iturin, fengycin or mixtures
thereof.
24. A composition of claim 23 wherein the biosurfactant is
surfactin.
25. A composition of claim 21 further comprising a microbial food
source.
26. Use of a composition of claim 21 in tertiary oil recovery.
27. Use of a composition of claim 21 as an antibacterial
composition to protect equipment from corrosion during tertiary oil
recovery or natural gas high pressure well processing.
28. Use according to claim 27 wherein the natural gas high pressure
well processing is water fracking.
29. Use of a composition of claim 21 as a fertilizer.
30. A method of tertiary oil recovery comprising i. treating an oil
well with a composition comprising a biosurfactant, and ii.
treating the oil well with a composition according to claim 21.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to a method of producing
biosurfactants, such as surfactin, comprising culturing at least
one biosurfactant-producing microbe in a liquid culture medium
comprising vinasse as a carbon source. Methods of using the crude
biosurfactant containing culture broth in tertiary oil recovery and
as antibacterial compositions in tertiary oil recovery are also
described. Methods of using the culture broth residue after
isolation of the biosurfactants as fertilizer and compositions for
this use are also described.
BACKGROUND OF THE INVENTION
[0002] There has been significant interest in the production of
biosurfactants, such as surfactin, as they are very powerful
surfactants being able to alter the interfacial tension of liquids
at very low concentrations. Furthermore, many biosurfactants have
antimicrobial activity.
[0003] Cyclic lipopeptides, such as surfactin, have a cyclic
peptide moiety and a moiety derived from a fatty acid. Surfactin
has a cyclic peptide of seven amino acids including both D- and
L-amino acids, Glu-Leu-D-Leu-Val-Asp-D-Leu-Leu, linked from the
N-terminus to the C-terminus to form a cyclic moiety by a
C.sub.12-C.sub.17 .beta.-hydroxy fatty acid as shown below.
[0004] Lichenysin has a similar structure with the amino acid
sequence differing from surfactin, Gln-Leu-D-Leu-Val-Asp-D-Leu-Ile,
linked from the N-terminus to the C-terminus to form a cyclic
moiety by a C.sub.12-C.sub.17 .beta.-hydroxy fatty acid.
[0005] Fengycin is a cyclic lipopeptide having the sequence
Glu-D-Orn-Tyr-D-Allo-Thr-Glu-D-Ala-Pro-Glu-D-Tyr-Ile where the
peptide is cyclized between the tyrosine phenoxy group of position
3 and the C-terminus of the Ile at position 10, the fatty acid is
attached to the peptide forming an amide with the N-terminus.
##STR00001##
[0006] Iturin refers to a group of cyclic peptides with the
sequence Asn-D-Tyr-D-Asn-Gln-Pro-D-Asn-Ser in which the N-terminus
and C-terminus are connected by a .beta.-amino fatty acid of
varying length.
[0007] Methods of producing these biosurfactants have focussed on
the identification of high yielding strains of Bacillus subtilis
[U.S. Pat. No. 3,030,789, Mulligan et al., 1989, Applied
Microbiology and Biotechnology 31:486-489], or by adjusting culture
conditions such as culturing in a magnetic field [JP-A-6-121668],
high iron concentrations [Wei et al., Enz. Microbial. Technol.
1989, 22:724-728], in the presence of peat [Sheppard et al. 1989,
Appl. Microbial. Biotechnol. 27:486-489] or reduced oxygen [Kim et
al., J. Ferment. Bioeng. 1927, 84:41-46].
[0008] In most cases, the culturing medium contains food products
or commodities as a catabolizable carbon source, for example,
glucose, maltose, sucrose, hydroylzed starch, molasses, potato
extract, malt, peat, vegetable oil, corn steep liquor, fructose,
syrup, sugar, liquid sugar, invert sugar, alcohol, organic acids
and their salts or alkanes [U.S. Pat. No. 7,011,959]. However, many
of these carbon sources are valuable commodities in other
commercial areas or are food products.
[0009] Vinasse is a by-product of the sugar industry. Sugar cane or
sugar beet is processed to product crystalline sugar, pulp and
molasses. The molasses is then processed by fermentation to produce
ethanol, ascorbic acid and other products. After the fermentation
and isolation of the desired product, the remaining residue is
vinasse. Vinasse is a waste product which is often disposed of by
burning [Cortez & Perez, Brazilian Journal of Chemical
Engineering, 1997, 14] or dumping into rivers.
[0010] Vinasse is a viscous, black-reddish liquid with total solids
content of 2-4% when obtained directly from sugar cane juice or
5-10% solids when obtained from molasses. It has a high biological
oxygen demand (BOD) (30 000-40 000) and high acidity (pH 4-5).
[0011] The dumping of Vinasse in rivers causes damage to aquatic
life because of its high BOD. Combustion is an expensive means of
disposal.
[0012] Vinasse has sometimes been used as a fertilizer. However,
its high acidity limits its usefulness to particular types of
soils.
[0013] Microbes and the biosurfactants they produce have been used
in tertiary oil recovery (microbial enhanced oil recovery, MEOR).
To enhance oil recovery from wells near the end of their
production, either [0014] i) compositions of nutrients that
stimulate endogenous bacteria to produce biosurfactants, break down
heavy oil to lighter oil, reduce oil viscosity, increase reservoir
(well) pressure and control mobility of oil during sweeping; [0015]
ii) or compositions of exogenous microbes that can perform these
functions in oil well conditions of high pressure, high temperature
and high salinity; [0016] have been used. However, a disadvantage
of MEOR is the biological production of hydrogen sulphide (also
referred to as souring), which may result in corrosion of piping
and machinery used in the recovery process.
[0017] Biosurfactants can be used to reduce interfacial tension
between the oil and rock surfaces in wells. The forces affecting
the flow of petroleum in porous rock reservoirs include gravity and
capillary pressure. Capillary pressure is a function of interfacial
tension between the oil/water and rock surface therefore, a
reduction in interfacial tension facilitates the flow of trapped
oil in the porous rock by reducing the coherent energy barrier at
the elastic interface layer of the two phases. The rock becomes
water-wet.
[0018] There is a need for methods of producing high yields of
biosurfactant while avoiding consumption of valuable commercial
and/or food commodities. There is also a need for effective means
of use of vinasse byproducts.
SUMMARY OF THE INVENTION
[0019] The present invention is predicated in part on the discovery
that vinasse can be used as a carbon source in the microbial
production of biosurfactants to achieve good yield at low cost.
[0020] In one aspect of the present invention there is provided a
method of producing biosurfactants comprising culturing at least
one biosurfactant-producing microbe in a liquid culture medium
comprising vinasse as a carbon source, wherein the culturing occurs
at pH 6 to 8 and a temperature of 25.degree. C. to 40.degree.
C.
[0021] In some embodiments, the at least one
biosurfactant-producing microbe is selected from Bacillus subtilis,
Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus
pumilus, Bacillus popilliae and mixtures thereof, especially
Bacillus subtilis subspecies subtilis NRRL B-3383 (US Department of
Agriculture, Agricultural Research Service, ARS Culture Collection
NRRL). In some embodiments, the at least one
biosurfactant-producing microbe is a mixture of Bacillus subtilis
and Bacillus licheniformis. In some embodiments, the vinasse is
sugar cane vinasse. In some embodiments, the vinasse is present in
the liquid culture medium in an amount of from 3% to 10% w/v. In
some embodiments, the biosurfactant produced is selected from
surfactin, lichenysin, fengycin, iturin and mixtures thereof,
especially surfactin, lichenysin and mixtures thereof, more
especially surfactin.
[0022] In some embodiments, the temperature of the culturing
process is 30.degree. C. to 35.degree. C. In some embodiments, the
pH is between 6.4 and 7.2. In some embodiments, the liquid culture
medium further comprises a catabolizable nitrogen source especially
salts of an ammonium ions and/or nitrate ions, such as ammonium
nitrate. In some embodiments, the liquid culture medium further
comprises at least one inorganic salt, such as salts of phosphates,
sulfates, iron, manganese, magnesium and calcium. In some
embodiments, the sulphate salts are minimised or omitted.
[0023] In some embodiments, the method further comprises aerating
the liquid culture medium during culturing and collecting foamate
produced during aeration. In some embodiments, aeration is begun
before inoculation of the culture medium. In some embodiments,
aeration is begun at the time of inoculation of the culture
medium.
[0024] In another aspect of the invention there is provided a
biosurfactant produced by the method described above, especially
where the biosurfactant is selected from surfactin, lichenysin and
mixtures thereof.
[0025] In some embodiments, the biosurfactant is produced in a
purity of at least 50%, especially at least 60%, 70%, 80% or above
90%, more especially at least 95%, for example, about 98%
purity.
[0026] In some embodiments, the biosurfactant is retained in the
culture broth. In other embodiments, the biosurfactant is isolated
from the culture broth.
[0027] In another aspect of the invention there is provided a
composition comprising at least one biosurfactant-producing microbe
and vinasse residue, wherein the vinasse residue is formed by
decomposition of vinasse by the at least one
biosurfactant-producing microbe during a fermentation process.
[0028] In some embodiments, the composition further comprises at
least one biosurfactant, such as surfactin, lichenysin, iturin,
fengycin or mixtures thereof, especially surfactin, lichenysin and
mixtures thereof, more especially surfactin.
[0029] In some embodiments, the composition contains trace amounts
of biosurfactant, for example, less than 30 .mu.mol of
biosurfactant. In other embodiments, the composition comprises
biosurfactant in the range of about 750 mg/L to 2000 mg/L.
[0030] In some embodiments, the composition further comprises an
added food source such as molasses, glycerine or the residue of
high fructose corn syrup.
[0031] In another aspect of the invention there is provided a use
of the compositions described above in tertiary oil recovery.
[0032] In another aspect of the invention there is provided a use
of the composition described above as an antibacterial composition
to protect equipment from corrosion during tertiary oil recovery or
natural gas high pressure well processing.
[0033] In yet another aspect of the invention there is provided a
use of the composition described above as a fertilizer.
DETAILED DESCRIPTION OF THE INVENTION
[0034] The articles "a" and "an" are used herein to refer to one or
to more than one (i.e. to at least one) of the grammatical object
of the article. By way of example, "an element" means one element
or more than one element.
[0035] Throughout this specification and the claims which follow,
unless the context requires otherwise, the word "comprise", and
variations such as "comprises" and "comprising", will be understood
to imply the inclusion of a stated integer or step or group of
integers or steps but not the exclusion of any other integer or
step or group of integers or steps.
[0036] In a first aspect of the invention there is provided a
method of producing a biosurfactant comprising culturing at least
one biosurfactant-producing microbe in a liquid culture medium
comprising vinasse as a carbon source, wherein the culturing occurs
at pH 6 to 8 and a temperature of 25.degree. C. to 40.degree.
C.
[0037] While the at least one biosurfactant-producing microbe may
be any microbe known to produce biosurfactants, in particular
embodiments, the at least one biosurfactant-producing microbe is
from the genus Bacillus, for example, they may be selected from
Bacillus subtilis, Bacillus licheniformis, Bacillus
amyloliquefaciens, Bacillus pumilus, Bacillus popilliae and
mixtures thereof. In some embodiments, one biosurfactant-producing
microbe is present in the liquid culture medium. In other
embodiments, two biosurfactant-producing microbes are present in
the liquid culture medium. In yet another embodiment, three
biosurfactant-producing microbes are present in the liquid culture
medium. In still further embodiments, four biosurfactant-producing
microbes are present in the liquid culture medium. In some
embodiments, the at least one biosurfactant-producing microbe is a
mixture of five biosurfactant-producing microbes. The at least one
biosurfactant-producing microbe may be a strain of microbe known to
produce biosurfactants in improved yields. For example, many
species of Bacillus produce biosurfactants, however, Bacillus
subtilis and Bacillus licheniformis are known to produce
significant quantities of biosurfactants. Furthermore, specific
strains of Bacillus subtilis are known to produce improved yields
of biosurfactants such as B. subtilis ATCC 21331, B. subtilis ATCC
21332, B. subtilis SD901 (FERM BP.7666), B. Subitilis NRRL B-3383
and B. subtilis RSA-203 or mixtures thereof. Many strains of
biosurfactant-producing microbes are commercially or publicly
available. In some embodiments, the at least one
biosurfactant-producing microbe is B. subtilis strain RSA-203.
[0038] RSA-203 is a microorganism that is a strain of Bacillus
subtilis. It is a rod-shaped, aerobic, Grain-positive,
.beta.-hemolytic microbe capable of forming endospores. Nucleic
acid sequence analysis confirms it is a strain of B. subtilis. A
sample of this microorganism was deposited at ATCC depository,
10801 University Boulevard, Manassas, Va. 20110-2209, United States
of America on 9 Jan. 2013, and has been allocated Accession No.
______.
[0039] RSA-203 produces significant amounts of the biosurfactant
surfactin. If culture conditions include foamate removal during
culture, surfactin may be produced in amounts of 250 mg/L to 1000
mg/L in the culture medium and 850 mg/L to 2 g/L in the
foamate.
[0040] In a particular embodiment, the at least one
biosurfactant-producing microbe is B. subtilis NRRL B-3383 which is
publicly available. In other particular embodiments, the at least
one biosurfactant-producing microbe is B. subtilis strain
RSA-203.
[0041] In some embodiments, the at least one
biosurfactant-producing microbe is a mixture of B. subtilis and B.
licheniformis. In other embodiments, the at least one
biosurfactant-producing microbe is a mixture of B. subtilis. B.
licheniformis, B. amyloliquefaciens, B. pumilus and Bacillus
popilliae. In these embodiments, the ratio of each microbe may be
adjusted to determine the amount of different biosurfactants
produced. In some embodiments, the B. subtilis is present in a
mixture of biosurfactant-producing microbes in about 50-98% of the
mixture, especially 60-95%, 70-95%, 80-95%, more especially about
90%.
[0042] The carbon source used in the liquid culture medium is
vinasse. Vinasse is a by-product of the sugar industry obtained
from the processing of sugar cane or sugar beet. The molasses
produced during sugar processing is fermented to produce ethanol
and ascorbic acid. The residue left after this fermentation is
referred to as vinasse. Vinasse is a viscous liquid with a total
solids content of 2-10%, high acidity pH 4-5 and high BOD (30
000-40 000).
[0043] In some embodiments, the amount of vinasse in the liquid
culture medium is from 3 to 20% w/v, especially 3 to 15% w/v, more
especially 3 to 12% w/v or 3 to 10% w/v, most especially about 10%
w/v. In some embodiments, the amount of vinasse is varied to obtain
a desired concentration of biosurfactant in the culture broth.
[0044] In some embodiments, a further carbon source is added in
addition to the vinasse. Suitable carbon sources include
carbohydrate sources such as molasses, dextrose, glucose, glycerine
and the like. The further carbon source may be present in the
liquid culture medium in an amount of 0 to 15% w/v especially 0 to
10% w/v.
[0045] In some embodiments, the culturing method takes place at a
sugar processing plant, for example, a sugar cane processing plant.
Advantageously, this reduces the costs involved in biosurfactant
production as if the vinasse is required to be transported to
another facility, the vinasse may need to be dehydrated to remove
excess water before transport and dehydration and transport costs
add to the cost of the biosurfactant.
[0046] The biosurfactant produced is preferably a cyclic
lipopeptide biosurfactant such as surfactin, lichenysin, iturin,
fengycin and mixtures thereof. Each of these biosurfactants may
contain mixtures of compounds varying in the chain length of the
fatty acid moiety of the lipopeptide. Addition of specific amino
acids and/or hydrocarbon fatty acids to the culture broth may
enable the production of biosurfactants with varying ratios of
lipid fatty acid chain lengths.
[0047] In some embodiments, the biosurfactant produced is selected
from surfactin and lichenysin and mixtures thereof. In other
embodiments, the biosurfactant produced is surfactin.
[0048] The temperature of the culturing process is 25.degree. C. to
40.degree. C., especially 30.degree. C. to 40.degree. C., more
especially about 30.degree. C. to 35.degree. C. The temperature
used may depend on the identity of the biosurfactant-producing
microbe. For example, the temperature is one that produces growth
of the microbes to a stress point which limits motility because of
the production of chemically produced microbial markers within the
broth. This allows for the maximum biosurfactant production for a
given microbial population. A person skilled in the art could
determine appropriate temperature for a given bacterial population
by routine trial methods.
[0049] The pH of the culture medium is maintained between 6 and 8,
especially 6 and 7.5, more especially 6.5 to 7.2. In a particular
embodiment, the culture medium is buffered at about pH 7 by
monobasic and dibasic phosphate buffer adjusted to pH 7 with
hydroxide such as sodium or potassium hydroxide.
[0050] The inoculum of at least one biosurfactant-producing microbe
is a culture of at least one biosurfactant-producing microbe in a
mid-log phase of growth. The inoculum is added to the culture
medium to provide an initial optical density at 600.sub.nm
(OD.sub.600nm) of 0.1 to 0.15.
[0051] In some embodiments the liquid culture medium further
comprises a catabolizable nitrogen source. In some embodiments, the
catabolizable nitrogen source is selected from a nitrogen
containing inorganic salt or nitrogen-containing organic compound
for example, ammonium salts, nitrate salts, urea, peptone, meat
extract, yeast extract, soybean cake, corn steep liquor, peptone,
or flour derived from legumes such as soybean, adzuki bean, pea,
broad bean, chick pea, lentil and string bean or extracts of such a
flour. In particular embodiments, the catabolizable nitrogen source
is an inorganic salt such as an ammonium salt or nitrate salt,
especially ammonium nitrate, ammonium chloride, ammonium acetate,
ammonium carbonate, ammonium bicarbonate, potassium nitrate, sodium
nitrate, magnesium nitrate, and calcium nitrate. In particular
embodiments, the catabolizable nitrogen source is ammonium
nitrate.
[0052] The amount of catabolizable nitrogen source present in the
liquid culture medium will depend on the nature of the source and
the availability of the nitrogen within the source. For example,
the nitrogen source may be present in an amount of 1 to 20 g/L.
When the nitrogen source is an inorganic nitrogen source, it may be
present in an amount of 1 to 10 g/L, especially 2 to 7 g/L, more
especially 3.5 to 4.5 g/L.
[0053] In some embodiments, the liquid culture medium further
comprises at least one inorganic salt, such as sulfates,
phosphates, chlorides, especially of metals such as manganese,
iron, sodium, potassium, magnesium and calcium. In some embodiments
the inorganic salts are selected from sulfates, chlorides, and
phosphates of ions such as manganese, sodium, potassium and iron or
mixtures of such salts. In a particular embodiment, the at least
one inorganic salt is selected from manganese sulfate, sodium
phosphate, calcium chloride, magnesium sulfate, ferrous sulfate and
mixtures thereof, especially sodium phosphate, manganese sulfate
and ferrous sulfate or mixtures thereof. In other embodiments, the
inorganic salts present are not sulfates. For example, in some
embodiments, the inorganic salts present are phosphates or
chlorides. This embodiment reduces the amount of sulfate present in
compositions that may be used in tertiary oil recovery where the
presence of sulfates may result in production of hydrogen
sulfide.
[0054] The inorganic salts vary in amount depending on the salts
used. If a source of phosphate is present, it may be present in an
amount of about 1 to 10 g/L, especially 2 to 7 g/L, more especially
4 to 7 g/L, most especially 5 to 6 g/L. Where inorganic salts are
added to provide trace elements such as iron, manganese, and
calcium, the amounts will vary between 1 mg/L and 5 g/L, for
example, calcium salts may be added in an amount of 0.5 g/L to 1
g/L, iron and manganese salts may be added in an amount of 1 to 10
mg/L, manganese salts may be added in an amount of 0.5 to 1 g/L,
magnesium salts may be added in an amount of 0.5 g/L to 5 g/L.
[0055] In some embodiments, the culture medium further comprises a
chelating agent. Particular chelating agents include amino
carboxylic acids and salts thereof, such as ethylene diamine
tetraacetic acid (EDTA), hydroxyethylethylenediamine triacetic
acid, 1,2-diamino-cyclohexane tetraacetic acid, ethylene
glycol-bis([beta]-aminoethyl ether)-N,N,N',N'-tetraacetic acid
(EGTA), diethylenetriamine-pentaacetic acid (DPTA),
triethylenetetraamine hexaacetic acid (TTG), aminodiacetic acid and
hydroxyethyl aminodiacetic acid. Particular chelating agents are
salts and mixed salts of EDTA such as dipotassium, ammonium,
calcium, disodium, trisodium and tetrasodium salts, most preferably
disodium or tetrasodium salts of EDTA, especially disodium EDTA.
The chelating agent is present in amount of between 0.1 and 5 mg/L,
especially 0.5 to 3 mg/L, more especially 1 to 2.5 mg/L of culture
medium.
[0056] The culturing method may occur on a small scale in
laboratory flasks in an incubator or may occur on larger scale,
such as industrial scale in a bioreactor. The method is conducted
under aerobic conditions.
[0057] The duration of the culturing process will depend on the use
of the culture broth. In some embodiments the culturing process has
a duration of 8 to 120 hours, especially 8 to 72 hours, 8 to 48
hours or 8 to 24 hours, for example 10 to 14 hours. The duration of
the culturing process is dependent on achieving a cell density
greater than OD.sub.600nm of .about.1.2 to 1.4, especially about
.about.1.3, where biosurfactant production begins and the length of
time taken to reach the stationary phase of growth,
.about.OD.sub.600nm of 1.8 to 2.5, where biosurfactant production
ceases.
[0058] In some embodiments, the process further comprises aeration
of the culture medium to provide dissolved oxygen. Typically, this
involves bubbling air through the culture medium at a rate of
between 1 L/minute to 3 L/minute, especially about 1.5 L/minute.
The rate of aeration may be readily determined by a person skilled
in the art. Aeration may occur from the beginning of the culturing
process or may begin after the culturing process has begun or may
begin prior to inoculation at the beginning of the culturing
process, especially from the beginning of the culturing process or
before inoculation. In particular embodiments, aeration maintains a
dissolved oxygen concentration of about 20 to 40%, especially 25 to
35%. In some embodiments, the dissolved oxygen concentration is
maintained at about 30% during the culturing process.
[0059] Once biosurfactant production has begun, the culture medium
may foam because of the presence of biosurfactant. In some
embodiments, the foam production may be controlled by spraying the
foamate with a mixture of alcohol such as ethanol, and solvent such
as dichloromethane or acetone. In some embodiments, the bioreactor
in which the fermentation is done is explosion proof. In some
embodiments, the foam collecting equipment is explosion proof. The
extent of pressure for which equipment must withstand is determined
by the pump pressure and flow rate into the foam column.
[0060] In some embodiments, the production of foamate is encouraged
and the foamate is collected from the culturing vessel. The foamate
comprises the biosurfactant produced together with small amounts of
culture medium. The foam may be collected via a rotary valve into a
tank with a slight vacuum or a tank with a spray column to break
the foam. The biosurfactant may be isolated from the foamate
collected. In some embodiments, the biosurfactant is isolated by
acidification followed by liquid/liquid extraction and then
evaporation of the liquids. In other embodiments, the biosurfactant
is isolated by centrifugation followed by liquid/liquid extraction
and evaporation or distillation of the liquids.
[0061] In some embodiments, the biosurfactant is isolated from the
culture broth after the culturing process is complete. For example,
the crude culture broth may be centrifuged to remove biomass. The
supernatant is then acidified to acidic pH, for example, pH 2 with
acid, such as HCl. The acidic pH results in the precipitation of
the biosurfactant, the acidified supernatant may be stood at
4.degree. C. for a period of time to ensure precipitation is
complete. The precipitate is then collected, for example, by
centrifugation or filtration and resuspended in water. The pH of
the suspension is adjusted to alkaline pH such as pH 8 to
solubilize the precipitate. The resulting aqueous solution may be
extracted with an organic solvent such as dichloromethane, ethyl
acetate, chloroform, especially dichloromethane, and the organic
phase evaporated to give the biosurfactant in high purity
crystalline form. In some embodiments, the biosurfactant may be
collected by foam distillation after culturing is complete.
[0062] The purified biosurfactant is suitable for many known uses
such as detergents, emulsifiers, wetting agents, dispersants,
solubilizing agents, antistatic agents, anti-clouding agents,
lubricants, pipe resistance lowering agents, or may be used in
cosmetics, foods, medical preparations, agricultural preparations,
inks and the like as known in the art.
[0063] In another aspect of the invention there is provided a
composition comprising at least one biosurfactant-producing microbe
and vinasse residue, wherein the vinasse residue is formed by
decomposition of vinasse by the at least one
biosurfactant-producing microbe during a culturing process.
[0064] In some embodiments, the composition is the crude culture
broth obtained from the method described above. In some
embodiments, the composition is depleted in biosurfactant as the
biosurfactant produced by the at least one biosurfactant-producing
microbe is removed during the culturing process by removal of
foamate comprising the biosurfactant. In these embodiments, the
composition may contain trace amounts of biosurfactant not removed
during the culturing process or produced by the at least one
biosurfactant-producing microbe after the culturing process has
been terminated. For example, in some embodiments, the amount is
less than 30 .mu.mol of biosurfactant.
[0065] This composition is useful as a fertilizer composition to
stimulate plant growth. The fertilizer may have a bacterial
population either from within the broth or added to the broth as a
symbiotic organism for plant root adhesion.
[0066] In other embodiments, the composition further comprises
biosurfactant, especially surfactin, lichenysin, iturin, fengycin
or mixtures thereof. This composition may be obtained by adding a
biosurfactant or mixture of biosurfactants to the composition or
may be obtained as the crude culture broth from the method above
from which no biosurfactant was isolated or only a portion of the
biosurfactant was extracted. Typically, the amount of biosurfactant
present in the composition is between 2 mg/L and 7000 mg/L, for
example 50 mg/L and 7000 mg/L or 500 mg/L, and 7000 mg/L, such as
500 mg/L and 3 g/L, especially 750 mg/L and 2 g/L.
[0067] In some embodiments where the composition comprises at least
one biosurfactant-producing microbe, vinasse residue and
biosurfactant, the composition may further comprise an added
microbial food source. In some embodiments, the added food source
is at least one selected from molasses, dextrose, glucose, vinasse,
glycerine and other carbohydrates. The addition of a food source
may allow the microbes to grow once they are in an appropriate
environment, for example, an oil well and thereby out compete other
undesirable microbes and produce additional biosurfactant that may
have an antimicrobial effect on undesirable microbes.
[0068] The composition comprising biosurfactant and optionally a
bacterial food source is useful in tertiary oil recovery, for
example, in microbial enhanced oil recovery (MEOR) processes and is
particularly useful in recovering oil from depleted calcium
carbonate rock reservoirs.
[0069] In some embodiments, an oil well is treated with a
biosurfactant composition containing a biosurfactant such as
surfactin in order to lower the surface tension of the oil and to
provide rock that is water-wet. The oil well may then subsequently
be treated with a composition of the invention.
[0070] In another aspect of the present invention there is provided
a method of tertiary oil recovery comprising [0071] 1. treating an
oil well with a composition comprising at least one biosurfactant,
and [0072] 2. treating the oil well with a composition comprising
at least one biosurfactant-producing microbe and vinasse residue,
wherein the vinasse residue is formed by decomposition of vinasse
by biosurfactant-producing microbes during a culturing process.
[0073] In some embodiments, the biosurfactant is in a composition
at a concentration of 2 mg/L to 4 g/L. In some embodiments, the
composition is a concentrate having about 1 g/L to 4 g/L
biosurfactant, especially about 1.5 g/L to 3 g/L, for example about
2 g/L. In other embodiments, the concentrate is diluted, for
example, with hydrofracking base water. For example, the
concentrate is mixed with hydrofracking base water as it is pumped
into the well. The concentrate may be diluted by an amount that
maintains at least 2 mg/L biosurfactant. For example dilution of
the biosurfactant composition may occur in the range of 1:100 to
1:2000, especially 1:1000 biosurfactant to diluent. A typical
concentrate comprises 10-40% w/v culture broth comprising microbes,
vinasse residue and crude biosurfactant, water and minerals, 10 to
20% w/v aqueous biosurfactant composition comprising 0.01 to 1% w/v
biosurfactant, 10 to 30% surfactant composition comprising 10 to
25% w/v surfactant and water, and the balance of the concentrate
being water. Upon dilution at entry into an oil well, the
composition may contain 94.9 to 98.98% w/v water, 1-5% w/v
surfactant, 0.01 to 1% w/v biosurfactant and 0.01 to 0.1% w/v
microorganisms. In some embodiments, the composition further
comprises a microbial food source, for example, a carbohydrate,
such as molasses, glucose, dextrose, vinasse and the like. The food
source may replace water in the concentrate up to about 30% w/v.
For example, a concentrate comprising a food source may comprise
10-40% w/v culture broth comprising microbes, vinasse residue and
crude biosurfactant, water and minerals, 10 to 20% w/v aqueous
biosurfactant composition comprising 0.01 to 1% w/v biosurfactant,
10 to 30% surfactant composition comprising 10 to 25% w/v
surfactant and water, 10% to 25% molasses optionally containing up
to 1% vinasse, and the balance of the concentrate being water.
[0074] The composition comprising the at least one
biosurfactant-producing microbe and vinasse residue is as described
above. In some embodiments, at least a portion of the microbes are
in spore form. In some embodiments, all of the microbes are in
spore form. In some embodiments, the spores are present in an
amount of 10.sup.2 cfu/mL to 10.sup.10 cfu/mL, especially 10.sup.4
cfu/mL to 10.sup.8 cfu/mL.
[0075] In some embodiments, steps 1 and 2 are performed separately.
In other embodiments, steps 1 and 2 are performed simultaneously
with a composition comprising at least one biosurfactant-producing
microbe and vinasse residue and at least one biosurfactant.
[0076] The composition comprising biosurfactant-producing bacteria,
vinasse residue and biosurfactant, is also useful as a biocide in
high salt content compositions, for example, 7% salt solution. Such
compositions are used as hydraulic fracturing (hydrofracking)
compositions in natural gas high pressure well processing. The
initial hydrofracking composition may not include high salt but
during use may solubilize salts from the rocks it contacts
increasing its salt concentration from between 0 and 12%.
[0077] The composition may be added to hydrofracking compositions
to prevent the formation of biofilms of unwanted bacteria such as
sulfate and iron reducing bacteria, on the inner surfaces of pipes
used in the natural gas processing. Steel pipes used in such
processing often suffer from corrosion by hydrogen sulfide
producing bacteria, or blockage or resistance on the inside of
piping by biofilms of other types of bacteria such as salt tolerant
bacteria. Problem bacteria include Acidithiobacillus ferrooxidans
and Desulfotomaculum halophilum.
[0078] Without wishing to be bound by theory, the composition of
the invention contains and produces biosurfactant and disrupts
biofilm formation by bacteria or disperses biofilms that have
already formed thereby reducing or preventing hydrogen sulphide
production and blockage or sludge forming on pipes. The
biosurfactant may also disrupt the cell walls of the unwanted
bacteria forming micelles and disrupting cell cytoplasm, resulting
in biocidal activity.
[0079] In some embodiments, particularly for use in hydrofracking
water, the biosurfactant-producing bacteria present are a
combination of B. subtilis and B. licheniformis and the
biosurfactants present are surfactin and lichenysin.
[0080] The invention will now be described with reference to the
following examples which illustrate some preferred aspects of the
invention. However, it is to be understood that the particularity
of the following description of the invention is not to supersede
the generality of the preceding description.
BRIEF DESCRIPTION OF THE FIGURE
[0081] FIG. 1 is a graphical representation showing the growth of
biosurfactant-producing microorganisms over time with no sulfate
ions or varying amounts of sulfate ions.
EXAMPLE 1
Production of Surfactin
[0082] Bacillus subtilis NRRL B-3383 strain (originally obtained
from the United States Department of Agriculture) from bacterial
culture on nutrient agar plates was transferred at a 2% volume by
volume inoculum into 4 L shake flasks containing 2.5 L of 10%
vinasse based MMS broth. The vinasse based MMS broth
containing:
TABLE-US-00001 component quantity Vinasse 100 mL Ammonium nitrate
4.1 g sodium phosphate dibasic 5.68 g tetrasodium tetrahydrate EDTA
1.8 mg Manganese sulfate 6.8 mg autoclaved deionized water to 1
L
[0083] The flasks were placed on orbital shakers (SKC 6100, Jeio
Tech) at 150 rpm while incubating at 30.degree. C. (MCO-801C
Incubator, Sanyo). After 72 hours, flasks were removed from the
incubator and the biomass removed from the crude culture broth by
centrifugation at 8,500 rpm for 20 min at 4.degree. C. (Sorvall
Evolution RC).
[0084] The pH of the resulting supernatant was brought to a pH of
2.0 using HCl which resulted in precipitation of surfactin and the
supernatant stored overnight at 4.degree. C. to ensure complete
precipitation. The precipitate was collected by centrifugation at
8,500 rpm for 20 minutes at 4.degree. C. Approximately 2.5 g/L of
crude material was collected in the pellet. The pellet was
suspended in deionized water and the pH adjusted to 8.0 using. 1 M
NaOH. The aqueous solution was extracted with an equal volume of
dichloromethane. The dichloromethane layer was separated and
allowed to evaporate to provide purified crystalline surfactin in
an amount of 50 mg/L to 750 mg/L.
[0085] The samples of crystalline surfactin were examined for
purity against a standard composition of pure surfactin (Sigma
Aldrich, 98% pure). Analysis of the standard composition by LC-MS
showed peaks with retention times at 1.03, 1.23, 1.61, 1.74, 2.15
and 2.93 minutes. Purity was calculated based on peak area.
[0086] Four samples tested for purity using the above method were
found to be 80%, 56%, 58% and 61% pure.
EXAMPLE 2
Production of Blends of Surfactin and Lichenysin
[0087] Bacillus subtilis and Bacillus licheniformis were used to
inoculate 4 L shake flasks containing 10% molasses based MMS broth.
The molasses based MMS broth containing:
TABLE-US-00002 component quantity molasses 100 mL Ammonium nitrate
4.1 g sodium phosphate dibasic 5.68 g tetrasodium tetrahydrate EDTA
1.8 mg Manganese sulfate 6.8 mg autoclaved deionized water to 1
L
[0088] The flasks were placed on orbital shakers (SKC 6100, Jeio
Tech) at 150 rpm while incubating at 30.degree. C. (MCO-801C
Incubator, Sanyo). After 72 hours, flasks were removed from the
incubator and the biomass removed from the culture broth by
centrifugation at 8,500 rpm for 20 min at 4.degree. C. (Sorvall
Evolution RC).
[0089] The crude products were labelled MEGR102, MEGR103 and
MEGR104, each being blends of varying concentrations of surfactin
and lichenysin.
EXAMPLE 3
Antibiotic Properties of MEGR102, MEGR103 and MEGR104
[0090] Each composition MEGR102, MEGR103 and MEGR104 was tested for
antibiotic properties against E. coli, Desulfotomaculum halophilum
and Acidithiobacillus ferrooxidans.
[0091] Each composition was tested by adding the composition to 7%
salt water containing 54,356 mg/L NaCl, 16,151 mg/L CaCl.sub.2,
2,383 mg/L MgCl.sub.2 and 535 mg/L KCl (to simulate hydrofracking
water) in amounts of 1 mg/L, 3 mg/L and 5 mg/L. The salt water
compositions of each concentration were then inoculated with E.
Coli (10.sup.8 cfu), Desulfotomaculum halophilum (10.sup.8 cfu) and
Acidithiobacillus ferrooxidans (10.sup.8 cfu).
[0092] The controls were inoculation of the 7% salt water with each
bacteria in equal amounts to the test samples without the addition
of MEGR composition.
[0093] The compositions were cultured at room temperature. At time
intervals, samples were taken and were analysed for culture growth
on agar plate to determine visual count of cfu.
[0094] The results are shown in the following Tables:
TABLE-US-00003 TABLE 1 MEGR102/E. coli Sample Time 0 2 hours 4
hours 6 hours 72 hours Control Growth Growth Growth Growth No
Growth 1 mg/L Growth Growth No Growth No Growth No Growth 3 mg/L
Growth Growth No Growth No Growth No Growth 5 mg/L Growth Growth No
Growth No Growth No Growth
TABLE-US-00004 TABLE 2 MEGR102/D. halophilum Sample Time 0 2 hours
4 hours 6 hours 72 hours Control Growth Growth Growth Growth Growth
1 mg/L Growth Growth No Growth No Growth No Growth 3 mg/L No No No
Growth No Growth No Growth Growth Growth 5 mg/L Growth No No Growth
No Growth No Growth Growth
TABLE-US-00005 TABLE 3 MEGR102/A. ferrooxidans Sample Time 0 2
hours 4 hours 6 hours 72 hours Control Growth Growth Growth Growth
Growth 1 mg/L Growth Growth Growth Growth No Growth 3 mg/L Growth
Growth Growth No Growth No Growth 5 mg/L Growth Growth No Growth No
Growth No Growth
TABLE-US-00006 TABLE 4 MEGR103/E. coli Sample Time 0 2 hours 4
hours 6 hours 72 hours Control Growth Growth Growth Growth 1 mg/L
Growth Growth Growth No Growth No Growth 3 mg/L Growth Growth No
Growth No Growth No Growth 5 mg/L Growth Growth No Growth No Growth
No Growth
TABLE-US-00007 TABLE 5 MEGR103/D. halophilum Sample Time 0 2 hours
4 hours 6 hours 72 hours Control Growth Growth Growth Growth Growth
1 mg/L Growth Growth Growth Growth Growth 3 mg/L Growth No No
Growth No Growth No Growth Growth 5 mg/L Growth No No Growth No
Growth No Growth Growth
TABLE-US-00008 TABLE 6 MEGR103/A. ferrooxidans Sample Time 0 2
hours 4 hours 6 hours 72 hours Control Growth Growth Growth Growth
Growth 1 mg/L Growth Growth Growth Growth No Growth 3 mg/L Growth
Growth Growth No Growth No Growth 5 mg/L Growth Growth No Growth No
Growth No Growth
TABLE-US-00009 TABLE 7 MEGR104/E. coli Sample Time 0 2 hours 4
hours 6 hours 72 hours Control Growth Growth Growth Growth No
Growth 1 mg/L Growth Growth No Growth No Growth No Growth 3 mg/L
Growth Growth No Growth No Growth No Growth 5 mg/L Growth No No
Growth No Growth No Growth Growth
TABLE-US-00010 TABLE 8 MEGR104/D. halophilum Sample Time 0 2 hours
4 hours 6 hours 72 hours Control Growth Growth Growth Growth Growth
1 mg/L Growth Growth No Growth No Growth Growth 3 mg/L Growth No No
Growth No Growth Growth Growth 5 mg/L No No No Growth No Growth No
Growth Growth Growth
TABLE-US-00011 TABLE 9 MEGR104/A. ferrooxidans Sample Time 0 2
hours 4 hours 6 hours 72 hours Control Growth Growth Growth Growth
Growth 1 mg/L Growth Growth Growth Growth Growth 3 mg/L Growth
Growth Growth No Growth Growth 5 mg/L Growth Growth Growth No
Growth No Growth
EXAMPLE 4
Reduction in Interfacial Tension of Oil
[0095] Using test method ASTM D1331-89 to measure interfacial
tension, the interfacial tension of 99.065 g of used bearing grease
was found to be 7510 dynes/cm.
[0096] The bearing grease was mixed with 25 mL of a composition
containing water 95.4%, surfactants 3.5%, dodecylbenzenesulfonic
acid >0.9% (DBSA), dextrose >0.5%, sodium hydroxide >0.2%,
Bacillus spores 10.sup.5 cfu/L and surfactin 5000 ppm, and allowed
to stand.
[0097] At 624 hours, the interfacial tension of the bearing grease
had reduced to 630 dynes/cm and at 696 hours, the interfacial
tension had reduced further to 420 dynes/cm.
[0098] The reduction in interfacial tension indicated the breakdown
of the bearing grease matrix to lower order hydrocarbons. This
process indicates the product is suitable for use in tertiary oil
recovery of heavy oil in carbonated rock formations.
EXAMPLE 5
The Effect of Sulfate Ions at Different Concentrations on
Culture
[0099] The effect of sulfate on culture broth was tested by
removing all sources of sulfate from the media and replacing them
with chloride salts. The culture broth contained monopotassium
phosphate/dipotassium phosphate buffer adjusted to pH 7 with
potassium hydroxide. Samples were then spiked with varying
concentrations of sodium sulfate (1.8 M) at 1 mL/L, 0.8 mL/L, 0.6
mL/L, 0.4 mL/L and 0.2 mL/L. Every half hour the optical density,
pH and surface tension was evaluated. This test was done with the
RSA-203 B. subtilis.
[0100] The results are shown in FIG. 1. The results show that the
microorganisms grow equally well with chloride salts as they do
with sulfate salts. In all samples, the surface tension had dropped
and stabilized around 27 dynes by 5 hours.
* * * * *