U.S. patent application number 10/052434 was filed with the patent office on 2002-05-16 for methods for purifying polyhydroxyalkanoates.
This patent application is currently assigned to Metabolix, Inc.. Invention is credited to Horowitz, Daniel M..
Application Number | 20020058316 10/052434 |
Document ID | / |
Family ID | 22460122 |
Filed Date | 2002-05-16 |
United States Patent
Application |
20020058316 |
Kind Code |
A1 |
Horowitz, Daniel M. |
May 16, 2002 |
Methods for purifying polyhydroxyalkanoates
Abstract
A method is provided for isolating and purifying PHA from
microbial or plant biomass that contains PHA. The method includes
the step of extracting PHA from the biomass using at least one
solvent while simultaneously subjecting the biomass to a filtration
process to remove cells. In a preferred embodiment of the method,
an aqueous slurry of the biomass is directly extracted by
diafiltration using an organic solvent. In a preferred
diafiltration process, an aqueous slurry of microbial cells
comprising PHA is recirculated through a filtration membrane,
wherein the membrane has a pore size sufficiently small to reject
individual cells or such aggregates of cells as may exist in the
slurry. As liquid is progressively removed from the biomass slurry
(by flowing out from the filtration membrane), an organic solvent,
preferably a water-miscible solvent that also is a solvent for the
PHA, is added to the biomass slurry at a rate which approximates
the rate of liquid permeation through the filter, thereby
maintaining the volume of the biomass slurry. Impurities which are
insoluble in water become dissolved in the solvent-water mixture
and pass through the filter membrane, and when the organic solvent
concentration reaches a certain level, the PHA becomes soluble and
flows through the filtration membrane and can be desolventized to
recover the polymer.
Inventors: |
Horowitz, Daniel M.;
(Alexandria, VA) |
Correspondence
Address: |
PATREA L. PABST
HOLLAND & KNIGHT LLP
SUITE 2000, ONE ATLANTIC CENTER
1201 WEST PEACHTREE STREET, N.E.
ATLANTA
GA
30309-3400
US
|
Assignee: |
Metabolix, Inc.
|
Family ID: |
22460122 |
Appl. No.: |
10/052434 |
Filed: |
January 18, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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10052434 |
Jan 18, 2002 |
|
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09570261 |
May 12, 2000 |
|
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60133747 |
May 12, 1999 |
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Current U.S.
Class: |
435/135 ;
528/274 |
Current CPC
Class: |
C08G 63/89 20130101;
C08G 63/90 20130101; C08G 63/06 20130101; C12P 7/625 20130101 |
Class at
Publication: |
435/135 ;
528/274 |
International
Class: |
C12P 007/62; C08G
063/90 |
Claims
I claim:
1. A method for isolating and purifying polyhydroxyalkanoates
(PHAs) derived from biomass comprising PRA, the method comprising
extracting PHA from the biomass using at least one solvent while
simultaneously subjecting the biomass to a filtration process to
remove cells.
2. The method of claim 1 wherein the filtration process comprises
diafiltration.
3. The method of claim 2 wherein the diafiltration is conducted at
a constant slurry volume.
4. The method of claim 1 wherein the biomass is subjected to a
gradient in solvent concentration.
5. The method of claim 1 wherein the biomass is derived from a
microbial source.
6. The method of claim 1 wherein the biomass is derived from a
plant or plant part.
7. The method of claim 6 wherein the plant is an oilseed plant.
8. The method of claim 1 wherein the biomass is provided as an
aqueous slurry and the solvent is an organic solvent.
9. The method of claim 8 wherein the organic solvent is
acetone.
10. The method of claim 8 wherein the aqueous slurry and organic
solvent form a solvent-water mixture, the method further comprising
gradually increasing the concentration of organic solvent in the
solvent-water mixture.
11. The method of claim 10 conducted in a diafiltration unit which
comprises a filter membrane wherein the concentration of organic
solvent is increased to cause impurities in the biomass which are
insoluble in water to dissolve in the solvent-water mixture and
pass through the filter membrane.
12. The method claim 11 wherein the concentration of organic
solvent is increased to cause the PHA to dissolve in the
solvent-water mixture and pass through the filter membrane to form
a PHA filtrate.
13. The method of claim 12 further comprising removing the solvent
from the PHA filtrate to recover the PHA.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] Priority is claimed to U.S. Ser. No. 60/133,747, filed May
12, 1999.
BACKGROUND OF THE INVENTION
[0002] The present invention generally relates to methods for
recovering polyhydroxyalkanoates ("PHAs") from microbial or plant
biomass. An improved understanding of the PHA biosynthetic pathways
has allowed for the use of microbial organisms, both natural and
recombinant, as well as plant cells, to produce significant
quantities of PHA. However, difficulties remain in developing
efficient and cost-effective recovery of the PHA at a useful levels
of quality and purity from these biological source materials.
Previous methods for isolating and purifying PHAs from biomass have
included, for example, aqueous routes as well as organic solvent
routes.
[0003] For example, U.S. Pat. No. 5,364,778 to Byrom discloses an
aqueous method wherein biomass comprising PHA is maintained as an
aqueous slurry in which the PHA is generally insoluble. The slurry
is subjected to various treatments designed to digest, degrade, or
otherwise make water-soluble the non-PHA biomass. This solubilized
biomass then is removed from the slurry by centrifugation,
filtration, or other means. Aqueous-based routes, however,
generally have certain disadvantages, particularly which applied to
large scale processing. Examples of these disadvantages include (a)
effective purification is made more difficult because many
impurities, including some surfactants useful for the solubilizing
treatments, may be tightly adsorbed to the surface of the PHA
particles; (b) many volumes (i.e. large quantities) of wash water
may be required by the process, creating used wash water and its
attendant disposal difficulties; (c) multiple solubilizing
treatments may be required to obtain high purity PHA; (d) drying of
the water-based PHA slurry may be time-consuming and costly; (e)
PHA particles may cause extensive fouling of filtration membranes,
centrifuges, and other process equipment; and (f) solubilizing
treatments may require expensive reagents and lengthy process times
and/or high temperatures to be effective.
[0004] Examples of organic solvent-based methods processes are
disclosed in U.S. Pat. No. 4,101,533 to Lafferty et al. and No.
5,422,257 to Ohleyer. In these methods, an organic solvent for the
PHA contained in a biomass is mixed with the biomass, resulting in
the dissolution of the PHA. The organic solution comprising the PHA
then is separated from the remaining insoluble biomass by
filtration, centrifugation, or other means. The organic solution
then is desolventized to recover the PHA. These organic solvent
routes suffer disadvantages similar to the disadvantages associated
with aqueous routes, including (a) a relatively large volume of
solvent is required to completely extract the PHA from biomass; (b)
biomass may need to dried prior to solvent extraction, which may be
costly and time-consuming; and (c) solvents may co-extract
impurities along with the PHA, such as lipids or other hydrophobic
biological materials, necessitating further processing of the
extract to obtain PHA of satisfactory purity. It would be
advantageous to develop improved, more cost-effective processes for
recovering PHA from PHA-containing biomass.
[0005] It is therefore an object of this invention to provide a
method of recovering PHA from PHA-containing biomass using a
process that is more simple, relatively faster, uses aqueous and/or
organic solvents more efficiently, and possibly yields a more pure
PHA product than conventional processes.
[0006] It is another object of the present invention to provide a
method of recovering PHA from PHA-containing biomass using a
process that can be employed economically in a commercial-scale
production process.
SUMMARY OF THE INVENTION
[0007] A method is provided for isolating and purifying PHA from
biomass which comprises PHA. The method includes the step of
extracting PHA from the biomass using at least one solvent while
simultaneously subjecting the biomass to a filtration process to
remove cells. In a preferred embodiment of the method, biomass
comprising PHA (for example an aqueous slurry of microbial cells
obtained from a fermentation process) is directly extracted by
diafiltration using an organic solvent, to obtain PHA.
[0008] In a preferred diafiltration process, an aqueous slurry of
microbial cells comprising PHA is recirculated through a filtration
membrane, wherein the membrane has a pore size sufficiently small
to reject individual cells or such aggregates of cells as may exist
in the slurry. An outflow of liquid from the filtration membrane
occurs under conditions where a pressure drop exists across the
filtration membrane. As the liquid is progressively removed from
the biomass slurry, an organic solvent, preferably a water-miscible
solvent that also is a solvent for the PHA, is added to the biomass
slurry. The solvent addition should be made at a rate which
approximates the rate of liquid permeation through the filter in
order to maintain the volume of the biomass slurry. As the
concentration of organic solvent in the slurry increases, various
impurities which are insoluble in water become dissolved in the
solvent-water mixture and pass through the filter membrane. When
the organic solvent concentration reaches a certain level, the PHA
becomes soluble and flows through the filtration membrane. The
filtrate comprising PHA then is desolventized to recover the
polymer.
[0009] The method has the advantages that (a) it is not generally
necessary to dry the biomass prior to solvent extraction; (b) it is
readily possible to fractionate the PHA from other impurities to
obtain relatively pure PHA in a single process, because the biomass
is subjected to a gradient in solvent concentration; (c) the entire
process of extracting and purifying PHA from biomass can be
accomplished using a minimum of process stages and equipment; and
(d) the method efficiently uses solvents, especially when the
biomass slurry is relatively concentrated and when the
diafiltration is conducted at a constant volume diafiltration.
Furthermore, by using volatile organic solvents, it is relatively
easy to desolventize the PHA solutions and to recover and reuse the
solvent from the filtrates generated in the diafiltration
process.
DETAILED DESCRIPTION OF THE INVENTION
[0010] A method has been developed for isolating and purifying
polyhydroxyalkanoates ("PHAs") from biomass comprising PHAs. The
method includes the step of extracting PHA from the biomass using
at least one solvent while simultaneously subjecting the biomass to
a filtration process to remove cells.
[0011] I. The PHA-Containing Biomass
[0012] The biomass materials are derived from PHA-producing plants
or PHA-producing microorganisms.
[0013] Polymer Compositions
[0014] As used herein, "polyhydroxyalkanoate" and "PHA" refer to
polymers that contain one or more units, for example between 10 and
100,000, and preferably between 100 and 30,000 units of the
following formula I:
--OCR.sup.1R.sup.2(CR.sup.3R.sup.4).sub.nCO--;
[0015] wherein n is an integer, for example between 1 and 15, and
in a preferred embodiment, between 1 and 4; and
[0016] wherein R.sup.1, R.sup.2, R.sup.3, and R.sup.4 independently
can be hydrocarbon radicals including long chain hydrocarbon
radicals; halo- and hydroxy-substituted radicals; hydroxy radicals;
halogen radicals; nitrogen-substituted radicals; oxygen-substituted
radicals; and/or hydrogen atoms.
[0017] As used herein, the formula --(CR.sup.3R.sup.4).sub.n-- is
defined as including the following formulas:
[0018] --CR.sup.3R.sup.4--(where n=1);
[0019] --CR.sup.3R.sup.4CR.sup.3'R.sup.4'--(where n=2); and
[0020] --CR.sup.3R.sup.4CR.sup.3'R.sup.4'CR.sup.3"R.sup.4"(where
n=3);
[0021] wherein R.sup.3, R.sup.4, R.sup.3', R.sup.4', R.sup.3", and
R.sup.4" can be independently hydrocarbon radicals including long
chain hydrocarbon radicals; halo- and hydroxy-substituted radicals;
hydroxy radicals; halogen radicals; nitrogen-substituted radicals;
oxygen-substituted radicals; and/or hydrogen atoms. Thus, formula I
includes units derived from 2-hydroxyacids (n=0), 3-hydroxyacids
(n=1),4-hydroxyacids (n=2), and 5-hydroxyacids (n=3), and
6-hydroxyacids (n=4).
[0022] These units may be the same in a homopolymer, or be more
different units, as for example in a copolymer or terpolymer. The
polymers typically have a molecular weight over 300, for example
between 300 and 10.sup.7, and in a preferred embodiment 10,000 to
10,000,000 Daltons.
[0023] Preferred PHAs include poly-3-hydroxyoctanoate (PHO) or
other microbial polyesters comprising hydroxyacids from C6 to C14
hydroxyacids. Other preferred polymers include
poly-3-hydroxybutyrate-co-3-hydroxyvaler- ate,
poly-3-hydroxybutyrate-co-3-hydroxypropionate,
poly-3-hydroxybutyrate-co-4-hydroxybutyrate,
poly-3-hydroxybutyrate-co-4-- hydroxyvalerate,
poly-3-hydroxybutyrate-co-3-hydroxyhexanoate,
poly-3-hydroxybutyrate-co-3hydroxyoctanoate,
poly-4-hydroxybutyrate, poly-3-hydroxypropionate,
poly-4-hydroxyvalerate.
[0024] Sources of PHA-Containing Biomass
[0025] The PHA biomass is typically generated from a fermentation
process (wherein the biological source is a microorganism which
naturally produces the PHAs or which can be induced to produce the
PHAs by manipulation of culture conditions and feedstocks, or
microorganisms) or produced in a plant, or plant part, which has
been genetically engineered so that it produces PHAs.
[0026] (i) Microbial Sources
[0027] Methods which can be used for producing PHA polymers from
microorganisms which naturally produce polyhydroxyalkanoates are
described in U.S. Pat. No. 4,910,145 to Holmes, et al.; Braunegg
et. al., J. Biotechnology 65:127-161 (1998).
[0028] Methods for producing PHAs in natural or genetically
engineered organisms are described in Madison & Huisman,
Microbiol. Mol. Biol. Rev. 63:1-53 (1999); Choi & Lee, Appl.
Microbiol Biotechnol. 51:13-21 (1999); Witholt & Kessler,
Current Opinion in Biotechnology 10:279-285 (1999); Williams &
Peoples, CHEMTECH, 26:38-44 (1996); U.S. Pat. Nos. 5,245,023;
5,250,430; 5,480,794; 5,512,669; 5,534,432 to Peoples and Sinskey;
and U.S. Pat. No. 5,563,239 to Hubbs et al. U.S. Pat. No. 5,292,860
to Shiotani et al. describes the manufacture of the PHA copolymer
poly(3-hydroxybutyrate-co-3-hydroxyhexanoate. U.S. Pat. No.
5,871,890 to Naylor describes the manufacture of PRAs by fermenting
Alcaligenes eutrophus on vegetable oil feedstocks.
[0029] (ii) Plant Sources
[0030] PHA can be recovered from essentially any plant type,
including transgenic plants which offers many advantages for the
production of PHAs.
[0031] Transgenic crop plants for production of PHAs can be
obtained using methods available in the art. (U.S. Pat. Nos.
5,245,023 and 5,250,430; 5,502,273; 5,534,432; 5,602,321;
5,610,041; PCT WO, 9100917, WO 9219747, WO 9302187, WO 9302194 and
WO 9412014; Poirier et al., 1992 Science 256:520-23, van der Leij
& Witholt, 1995, Can. J Microbiol. 41 (supp):222-38; Nawrath
& Poirier, 1996, presented at The International; Symposium on
Bacterial Polyhydroxyalkanoates, Eggink et al., eds. Davos
Switzerland, August 18-23; Williams & Peoples, 1996, CHEMTECH
26:38-44). Transgenic plant crop production can produce PHA
polymers at both a price and a scale that is competitive with
petrochemical derived plastics. Transgenic plant derived PHA
polymers or their derivatives can be processed and separated from
plant biomass in commercially usefuil forms. The location of the
PHA in the plant crop can be varied to maximize the yield of PHA
from the plant. For example, the plants can be monocots or dicots
and suitable plant source materials can be derived from roots,
stems, leaves, flowers, fruits, and seeds.
[0032] PHAs can be isolated from plant biomass derived from plants
such as soybean, cotton, coconuts, groundnuts, rapeseed, sunflower
seed, olive, palm, sesame seed, linseed, castor, safflower seed,
tobacco, sugarcane, swithchgrass, and potato. In addition to the
PHA polymers, the plant oil in seed crop plants can be isolated and
recovered during the processing, as described in PCT WO 97/15681 to
Metabolix, Inc. and U.S. Ser. No. 08/548,840, which is incorporated
by reference herein. The methods for processing the plant biomass
can be tailored based on the properties of the particular PHA
polymer or derivative being isolated, and based on the type of
plant crop and the plant components being extracted.
[0033] III. Process for PHA Recovery from Biomass
[0034] The method includes the step of extracting PHA from the
biomass using at least one solvent while simultaneously subjecting
the biomass to a filtration process to remove cells.
[0035] Diafiltration
[0036] In a preferred embodiment of the method, biomass comprising
PHA (for example an aqueous slurry of microbial cells obtained from
a fermentation process) is directly extracted by modification of a
typical diafiltration process in which an organic solvent is used
instead of an aqueous diluent. Standard diafiltration processes are
well known in the art and are described for example by Zeman &
Zydney, Microfiltration and Ultrafiltration Principles and
Applications, Marcel Dekker, Inc. New York, N.Y. pp. 391-96 (1996).
During this modified process, as the concentration of organic
solvent increases, the PHA is solubilized and appears in the eluant
which is collected. The PHA is then recovered from the eluant by
standard procedures including precipitation in a non-solvent,
solvent evaporation or stripping to recover the PHA. The solvent
containing eluant is retained and the solvent recovered by
distillation or other techniques well known in the art.
[0037] In a preferred embodiment of the method, biomass comprising
PHA (for example an aqueous slurry of microbial cells obtained from
a fermentation process) is directly extracted by diafiltration
using an organic solvent, to obtain PHA.
[0038] The method has the advantages that (a) it is not generally
necessary to dry the biomass prior to solvent extraction; (b) it is
readily possible to fractionate the PHA from other impurities to
obtain relatively pure PHA in a single process, because the biomass
is subjected to a gradient in solvent concentration; (c) the entire
process of extracting and purifying PHA from biomass can be
accomplished using a minimum of process stages and equipment; and
(d) the method efficiently uses solvents, especially when the
biomass slurry is relatively concentrated and when the
diafiltration is conducted at a constant slurry volume ("constant
volume diafiltration"). Furthermore, by using volatile organic
solvents, it is relatively easy to desolventize the PHA solutions
and to recover and reuse the solvent from the filtrates generated
in the diafiltration process.
[0039] In a preferred diafiltration process, an aqueous slurry of
microbial cells comprising PHA is recirculated through a filtration
membrane, wherein the membrane has a pore size sufficiently small
to reject individual cells or such aggregates of cells as may exist
in the slurry. An outflow of liquid, the eluant which can be an
aqueous solution, an aqueous solution/miscible solvent mixture, or
solvent, from the filtration membrane occurs under conditions where
a pressure drop exists across the filtration membrane. As the
liquid is progressively removed from the biomass slurry, an organic
solvent, preferably a water-miscible solvent that also is a solvent
for the PHA, is added to the biomass slurry. The solvent addition
should be made at a rate which approximates the rate of liquid
permeation through the filter in order to maintain the volume of
the biomass slurry. As the concentration of organic solvent in the
slurry increases, various impurities which are insoluble in water
become dissolved in the solvent-water mixture and pass through the
filter membrane. When the organic solvent concentration reaches a
certain level, the PHA becomes soluble and flows through the
filtration membrane. The filtrate comprising PHA then is
desolventized to recover the polymer.
[0040] Organic Solvents and Solvent Recovery
[0041] Solvents suitable for extracting the PHA from the biomass
are any water miscible solvent capable of extracting the PHA. It is
well known in the art which solvents are suitable for extracting
the different PHA polymer compositions as described for example in
U.S. Pat. Nos. 5,821,299 and 5,942,597 to Noda; U.S. Pat. No.
6,043,063 to Kurdikar; and PCT WO 97/15681 to Metabolix, Inc., all
of which are incorporated herein by reference.
[0042] A preferred organic solvent for PHAs such as
poly-3-hydroxyoctanoate (PHO) or other microbial polyesters
comprising hydroxyacids from C6 to C14 in length is acetone.
Acetone is also suitable for extracting
poly-3-hydroxybutyrate-co-4-hydroxybutyrate. Other ketones and
alcohols, especially alcohols above C2, can be used as described
above. For PHO, solubilization of the polyester typically occurs at
an acetone concentration from 85-98% in water (volume basis).
[0043] Organic solvents useful in the methods described herein
include both halogentated and nonhalogentated solvents.
Representative examples include solvents selected from cyclic and
acyclic (linear and branched) R'--OH alcohols where
R'.dbd.C.sub.4-C.sub.10, cyclic and acyclic R"--COOR'" esters where
R".dbd.H or C.sub.1-C.sub.6 and R'".dbd.C.sub.1-C.sub.7, cyclic and
acyclic R"--COOR'" esters where R".dbd.H or C.sub.1-C.sub.6 and
R'".dbd.C.sub.1-C.sub.7 and wherein at least one oxygen is
substituted for at least one carbon in R" or R'", cyclic and
acyclic R.sup.1--CON--(R.sup.2).sub.2 amides where R.sup.1.dbd.H or
C.sub.1-C.sub.6 and R.sup.2.dbd.C.sub.1-C.sub.6, and cyclic and
acyclic R.sup.3--CO--R.sup.4 ketones where
R.sup.3.dbd.C.sub.1-C.sub.6 and R.sup.4.dbd.C.sub.1-C.sub.6.
[0044] Specific examples include acetone, butyl acetate, isobutyl
acetate, ethyl lactate, isoamyl acetate, benzyl acetate, 2-methoxy
ethyl acetate, tetrahydrofurfuryl acetate, propyl propionate, butyl
propionate, pentyl propionate, butyl butyrate, isobutyl
isobutyrate, ethyl butyrate, ethyl valerate, methyl valerate,
benzyl benzoate, methyl benzoate, dimethyl succinate, dimethyl
glutarate, dimethyl adipate, isobutyl alcohol, 1-butanol,
2-methyl-1-butanol, 3methyl-1butanol, 1-pentanol, 3-pentanol, amyl
alcohol, allyl alcohol, hexanol, heptanol, octanol, cyclohexanol,
2-ethylhexanol, tetrahydrofurfuryl alcohol, furfuryl alcohol,
benzyl alcohol, 2-furaldehyde, methyl isobutyl ketone, methyl ethyl
ketone, g-butyrolactone, methyl n-amyl ketone, 5-methyl-2-hexanone,
ethyl benzene, 1,3-dimethoxybenzene, cumene, benzaldehyde,
1,2-propanediol, 1,2-diaminopropane, ethylene glycol diethyl ether,
1,2,3-trimethylbenzene, 1,2,4-trimethylbenzene, 1,3-dioxane,
1,4-dioxane, 1-nitropropane, toluene-2,4-diisocyanate, acetic acid,
acrylic acid, acetic anhydride, alpha-methylstyrene, acetophenone,
toluene, ethylene glycol diacetate, dimethyl sulfoxide, dimethyl
acetamide, dimethyl formamide and propylene carbonate.
[0045] Solvents which can be used include solvents or solvent
mixtures including chloronated organic solvents such as chloroform,
methylene chloride, dichloroethane, trichloroethane,
tetrachloroethane and dichloroacetate. For example, hydrocarbon
stabilized chloroform can be used. Other solvents which have been
used to extract PHAs from microbial sources which may be used
include alkyl carbonates, such as propylene carbonate and ethylene
carbonate, trifluoroethanol, acetic anhydride, dimethylformamide,
ethylacetoacetate, triolein, toluene, dioxane, tetrahydrofuran,
diethylether, pyridine, hydroxyacids and alcohols having more than
3 carbon atoms, as well as mixtures thereof.
[0046] Solvent recovery can be carried out by processes well known
to those skilled in the art and includes distillation or extraction
into a second solvent or solvent mixture which is not miscible with
water and subsequent separation by distillation
[0047] Recovery of the PHA From the Eluant or Filtrate
[0048] Once the polymer appears in the filtrate or eluant, it is
necessary to recover the polymer from the solvent and also to
recover the solvent. Techniques for doing this are also well known
in the art and include solvent stripping or evaporation, steam
stripping or solvent precipitation with a non-solvent.
[0049] The compositions and methods described herein will be
further understood with reference to the following non-limiting
examples.
Example 1
[0050] Typical Production of PHA-Containing Microbial Biomass
[0051] Pseudomonas species bacteria were fermented as follows to
produce PHA. Octanoic acid (Prifrac 2901) was obtained from
Unichema International, Chicago, Ill.; all other chemicals were
reagent grade. Medium A contained deionized water plus the
following (per L final volume): Octanoic acid (2.16 g),
NaNH.sub.4HPO.sub.4 (3.8 g), K.sub.2HPO.sub.4 (5.7 g),
KH.sub.2PO.sub.4 (3.7 g), MgSO.sub.4 (0.12 g), CaCl.sub.2 (20 mg),
FeSO.sub.4.7H.sub.2O (40 mg), MnSO.sub.4.H.sub.2O (10 mg),
CoCl.sub.2.6H.sub.2O (4.5 mg), ZnSO.sub.4-7H.sub.2O (2 mg),
Na.sub.2MoO.sub.4.2H.sub.2O (2 mg), CuCl.sub.2.2H.sub.2O (1 mg),
Al.sub.2(SO.sub.4)3.16H.sub.2O (1.3 mg), H.sub.3BO.sub.4 (465
.mu.g), NiSO.sub.4.6H.sub.2O (180 .mu.g), corn steep liquor (Sigma,
0.5 mL). All components with the exception of octanoic acid were
sterilized by heating (121.5.degree. C.) or filtration and
transferred aseptically into the vessel. The pH was adjusted to 6.7
and maintained throughout all fermentations at that value (.+-.0.1
pH unit). Control of pH was effected using 30% (wt/wt) aqueous
ammonia and 85% (wt/wt) phosphoric acid, which were added as needed
via an automatic pH controller. Antifoam agent (Breox FMT30
obtained from Inspec Group, Southampton, UK) was added as needed
during fermentations.
[0052] The culture was fed as described below with defined medium
doses under metabolic ([DO]) control. Each defined medium dose
consisted of three subdoses which were added simultaneously into
the culture through separate addition ports. Subdose #1 consisted
of octanoic acid (1.46 g per L initial culture volume); subdose #2
consisted of 30% (wt/wt) aqueous ammonia (0.36 g per L initial
culture volume); subdose #3 consisted of 0.3M MgSO.sub.4 (0.4 mL
per L initial culture volume). The time required to provide a
single defined medium dose into the fermenter was approximately two
minutes. Each defined medium dose provided sufficient nutrition to
generate about 1.3 g/L total solids.
[0053] Pseudomonas putida was stored in frozen culture and
propagated on 1.5% agar plates (Medium A). Frozen cultures were
thawed, plated, and grown for 48 hr. at 30.degree. C. Single
colonies were then replated and grown for 24 hr. at 30.degree. C.
Single colonies were then chosen and transferred into liquid medium
A (1 L) and grown in a shaker at 30.degree. C. for 24 hr. This seed
culture was then transferred into a 150 L fermenter containing
defined Medium A (60 L) at 30.degree. C. The fermenter was equipped
with a single [DO] probe and pH probe. The culture was fermented at
30.degree.C. with agitation (impeller speed 150-600 rpm) and
aeration with atmospheric air (60 L/min) under a head pressure of 3
psi (20.7 MPa). Agitation rate was increased progressively through
the course of the fermentation. Dissolved oxygen concentration was
monitored continuously and a defined medium dose was provided in
response to each sustained (>10 sec), significant (>10%
saturation increase above prevailing baseline) increase in measured
[DO]. During the first 8-9 hr. of fermentation, the [DO] dropped
steadily from 100% to ca. 0% saturation. Thereafter the [DO]
maintained a baseline condition of ca. 0% saturation. Agitation and
aeration rates were controlled within these ranges to try to
maintain [DO] =1% saturation. Sustained increases in the [DO] above
10% saturation were considered the result of carbon source
exhaustion and triggered the automatic addition of a defined medium
dose. Addition of each dose resulted in a decrease in [DO] back to
the baseline condition. However, a feedback mechanism prevented
multiple defined medium dose additions in case the [DO] were slow
to return below 10% saturation. A total of 24 defined medium doses
were provided over the course of the 21 hr. fermentation.
[0054] Immediately at the conclusion of the above fermentation, the
culture was transferred aseptically into a 1500 L fermenter
containing 640 L of defined Medium A. The fermenter was equipped
with a single [DO] probe and pH probe, and a mass spectral off-gas
analyzer. Fermentation was conducted under conditions similar to
those above, with a temperature of 30.degree. C., agitation
(impeller speed 60-210 rpm), and aeration with atmospheric air
(600-950 L/min) under a head pressure of 3 psi (20.7 MPa). After
inoculation, [DO] dropped rapidly (within 3 hr.) to near 0%
saturation. Agitation and aeration rates were controlled within the
aforementioned ranges to try to maintain [DO]=1% saturation. The
fermentation was continued for 41 hr.
[0055] The final culture consisted of 750 L containing 116.7 g/L of
dry solids, of which 66.3% was PHA. The PHA had the following
monomeric composition: R-3-hydroxyhexanoic acid (10%),
R-3-hydroxyoctanoic acid (88%), R-3-hydroxydecanoic acid (2%). The
isolated polymer showed M.sub.w=115,000; M.sub.n=70,000 (GPC in
CHCl.sub.3); T.sub.m=50.degree. C.; and T.sub.g=-38.degree. C.
Example 2
[0056] Recovery of PHA from Microbial Biomass
[0057] A cell slurry containing polyhydroxyalkanoate (PHA) was
processed as follows to obtain a purified polymer. Cells of
Pseudomonas sp. bacteria were fermented as described in Example 1
on a commercial mixture comprising principally octanoic and
decanoic acids (C810 Fatty Acid, Procter & Gamble, composition
56% C8, 39% C10, balance other fatty acids). The initial slurry (5
L), which comprised approximately 13% (wt/wt) suspended solids, was
centrifuged at 4000 g for 20 min. The pellet fraction was
resuspended to its original volume in deionized water and then
recentrifuged under identical conditions. The pellet fraction was
then resuspended in acetone to its original volume. This slurry
comprising cellular material, water, and acetone (total
solids=12.8% wt/wt) was then processed using the experimental
apparatus described below.
[0058] The experimental microfiltration apparatus comprised an
explosion proof, variable speed, eccentric screw pump (Allweiler)
capable of at least 15 L/min flow against a head pressure of 0.6
MPa; a stainless steel and polypropylene piping circuit; and a
housing containing an alumina ceramic tubular microfiltration
element (U.S. Filter Membralox 1T1-70, 0.5 .mu.m nominal cutoff,
0.0055 m.sup.2 membrane area). In addition the apparatus was
equipped with pressure gauges, temperature probes, a ball valve for
pressure regulation, and a 20-L covered slurry tank. The liquid
level in the slurry tank was maintained approximately constant
through continuous addition of acetone via an adjustable feeding
pump. During operation, the slurry was continuously circulated
through the tubular ceramic membrane at a cross flow of
approximately 15 L/min and an average transmembrane pressure of
0.3-0.6 MPa. Transmembrane flow rate ranged from 8-30 mL/min
(90-330 L/m.sup.2/hr). The system temperature was maintained at
20-32.degree. C. by means of a glycol/water cooling jacket
installed on the pump head. As the result of the continuous removal
of permeate and continuous addition of pure acetone, the
concentration of acetone in the slurry increased throughout the
operation.
[0059] The acetone/water-comprising permeate was collected in a
series of fractions. When the ratio of acetone to water in the
slurry exceed a critical value of approximately 9:1 (wt/wt), the
PHA copolymer became soluble and was passed through the ceramic
membrane. The concentration of polymer in the permeate peaked at
5.8% wt/wt. The permeate was collected until the concentration of
solids was <0.1% (wt/wt). Fractions containing PHA were combined
(14 L), and the polymer was precipitated by addition of 10%
(vol/vol) deionized water. The filter concentrate comprised
acetone, water, and essentially PHA-free cell debris.
[0060] Modifications and variations of the present invention will
be obvious to those of skill in the art from the foregoing detailed
description. Such modifications and variations are intended to come
within the scope of the following claims.
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