U.S. patent application number 16/848430 was filed with the patent office on 2021-01-07 for process of separating components of a fermentation broth.
The applicant listed for this patent is Genomatica, Inc.. Invention is credited to Mark J. Burk, Warren Clark, Michael Japs.
Application Number | 20210002194 16/848430 |
Document ID | / |
Family ID | |
Filed Date | 2021-01-07 |
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United States Patent
Application |
20210002194 |
Kind Code |
A1 |
Clark; Warren ; et
al. |
January 7, 2021 |
PROCESS OF SEPARATING COMPONENTS OF A FERMENTATION BROTH
Abstract
A process of isolating 1,4-butanediol (1,4-BDO) from a
fermentation broth includes separating a liquid fraction enriched
in 1,4-BDO from a solid fraction comprising cells, removing water
from said liquid fraction, removing salts from said liquid
fraction, and purifying 1,4-BDO. A process for producing 1,4-BDO
includes culturing a 1,4-BDO-producing microorganism in a fermentor
for a sufficient period of time to produce 1,4-BDO. The
1,4-BDO-producing microorganism includes a microorganism having a
1,4-BDO pathway having one or more exogenous genes encoding a
1,4-BDO pathway enzyme and/or one or more gene disruptions. The
process for producing 1,4-BDO further includes isolating
1,4-BDO.
Inventors: |
Clark; Warren; (Yale,
OK) ; Japs; Michael; (San Diego, CA) ; Burk;
Mark J.; (San Diego, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Genomatica, Inc. |
San Diego |
CA |
US |
|
|
Appl. No.: |
16/848430 |
Filed: |
April 14, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15975630 |
May 9, 2018 |
10662136 |
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16848430 |
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14066598 |
Oct 29, 2013 |
9994505 |
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15975630 |
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12793623 |
Jun 3, 2010 |
8597918 |
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14066598 |
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61184292 |
Jun 4, 2009 |
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Current U.S.
Class: |
1/1 |
International
Class: |
C07C 29/76 20060101
C07C029/76; C12P 7/18 20060101 C12P007/18 |
Claims
1-31. (canceled)
32. A process of isolating 1,4-BDO from a fermentation broth
comprising removing a portion of solids by disc stack
centrifugation to provide a liquid fraction, removing a further
portion of solids from the liquid fraction by ultrafiltration,
removing a portion of salts from the liquid fraction by
nanofiltration, removing a further portion of salts from the liquid
fraction by ion exchange, evaporating a portion of water, and
distilling 1,4-BDO.
33. The process of claim 32, wherein the step of removing a further
portion of salts by ion exchange comprises passing the liquid
fraction through an ion exchange column selected from the group
consisting of a cation exchange column, an anion exchange column, a
mixed-bed ion exchange column, and combinations thereof.
34. The process of claim 32, wherein the step of evaporating a
portion of water comprises passing the liquid fraction through an
evaporator system, said evaporator system comprising an effect
selected from the group consisting of a falling film evaporator, a
short path falling film evaporator, a forced circulation
evaporator, a plate evaporator, a circulation evaporator, a
fluidized bed evaporator, a rising film evaporator, a
counterflow-trickle evaporator, a stirrer evaporator, a spiral tube
evaporator, and combinations thereof, said evaporator system
optionally comprising a recompression system comprising a
recompressor selected from the group consisting of a thermal
recompressor, a mechanical recompressor, and combinations
thereof.
35. (canceled)
Description
[0001] This application is a continuation under 35 U.S.C. .sctn.
120 of U.S. patent application Ser. No. 15/975,630, filed May 9,
2018, which is a continuation under 35 U.S.C. .sctn. 120 of U.S.
patent application Ser. No. 14/066,598, filed Oct. 29, 2013, now
U.S. Pat. No. 9,994,505, which is a continuation under 35 U.S.C.
.sctn. 120 of U.S. patent application Ser. No. 12/793,623, filed
Jun. 3, 2010, now U.S. Pat. No. 8,597,918, which claims the benefit
of priority of U.S. Provisional Application Ser. No. 61/184,292,
filed Jun. 4, 2009, the entire contents of each of which are
incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] This invention relates generally to the separation of
components of a fermentation broth and, more specifically to the
isolation of water miscible compounds having boiling points higher
than water from other fermentation broth components.
[0003] Environmental and cost reduction incentives exist to design
process schemes that have the ability to separate and optionally
recycle components in a fermentation including the cell mass,
residual media and media salts, residual substrate such as sucrose
and/or glucose, and water. Efforts have also been made to recycle
cell mass as a means to improve the fermentation productivity. Less
effort has been made in the area of recovering the residual media
and media salts for reuse in the fermentation. In this regard, most
efforts have focused on reducing initial media costs, rather than
downstream recovery. The resulting "low cost" media is often not
optimal for cell growth and product production. By developing
effective methods for the recovery of media components, a more
optimal media recipe can be utilized with fewer restrictions on
initial raw material costs.
[0004] The isolation of compounds on large scale with useful purity
is a complex challenge in process chemistry. Differences in scale
alone can render isolation procedures developed on laboratory
benchtop scale impractical or even not viable at pilot or
commercial scales. Isolation of compounds from complex mixtures
depends on numerous factors including whether the compound is a
solid or liquid at ambient temperatures, the compounds boiling
point, density, polarity, the presence or absence of pH sensitive
functional groups, and solubility in organic solvents versus water.
These factors also apply to all other components of the mixture
from which the compound of interest is to be isolated. Another
property that factors into isolation of a compound, organic
compounds in particular, is how it partitions between two
immiscible phases, such as between water and an organic solvent.
Compounds that are particularly polar are often more soluble in
water than in common organic solvents used in extraction processes.
Some compounds are particularly challenging to isolate from water
by extractive methods due to their amphiphilic character.
Amphiphiles are compounds that possess both a polar portion and a
lipophilic portion. These compounds can complicate isolation by
extraction by causing intractable emulsions.
[0005] Moreover, when a compound is prepared from a fermentation
the amount of water can be substantially higher than the compound
of interest, requiring isolation of a minor component from a
complex mixture. Isolation of compounds that boil at a higher
temperature than water further adds to the complexity and cost of
the separation since the compound cannot be distilled directly from
the fermentation broth as is the case, for example, in an ethanol
fermentation process. In this regard, interactions between the
compound of interest and water can cause the two entities to
co-distill as an azeotrope at a boiling point different from the
two purified components. Azeotrope formation is not readily
predictable. This can diminish recovery of the compound of interest
when trying to separate it from water. When a compound has polar
functional groups another concern is how it may interact with other
compounds present in the water phase, including any salts and metal
ions, for example.
[0006] The nature of the functional groups present in a compound of
interest can complicate the separation of salts. For example, one
or more functional groups of a compound can interact with or
chelate cations or anions. Chelation occurs in a size dependent
manner with respect to the cation or anion and is also dependent on
the disposition of the functional groups on the compound of
interest. Chelation and other interactions can render some salts
soluble in a liquid compound even in the absence of water, while
other salts can be insoluble in the absence of water despite the
presence of a compound with functional groups capable of
interacting with salts. These types of effects on salt solubility
are difficult to predict. Further adding to the complexity of the
interaction between a compound and salts, is the nature of any
co-solvents. For example, during the isolation of a compound of
interest that is water miscible, hydrogen bonding and other
interactions with water can disrupt the interaction between the
salts and the compound of interest. Thus, in some cases a salt can
be separated more readily from a compound in the presence of some
amount of water. However, the amount of water that balances salt
supersaturation allowing salt separation by crystallization, for
example, while maintaining water's ability to disrupt chelation and
other interactions between a compound of interest and any salts is
difficult to predict.
[0007] Yet a further challenge in developing isolation methods is
the potential reactivity of biosynthetic byproducts such as organic
acids, excess substrate, and the like. Under conditions of heating,
excess substrate can degrade and cause undesirable coloration of
product. Additionally, some byproducts can react with the product
of interest, effectively lowering isolation yields. These
byproducts can include those formed during fermentation as well as
byproducts formed during steps of the isolation procedure itself,
for example due to degradation processes at elevated temperatures
during a distillation, water evaporation, and the like.
[0008] Thus, there is a need to develop processes that allow for
the isolation of water miscible compounds that have boiling points
higher than water from microbial fermentations, while bearing in
mind the environmental and cost benefit of recycling other
fermentation components. The present invention satisfies this need
and provides related advantages as well.
SUMMARY OF THE INVENTION
[0009] In some aspects, embodiments disclosed herein relate to
process of isolating 1,4-butanediol (1,4-BDO) from a fermentation
broth that includes separating a liquid fraction enriched in
1,4-BDO from a solid fraction comprising cells, removing water from
said liquid fraction, removing salts from said liquid fraction, and
purifying 1,4-BDO.
[0010] In other aspects, embodiments disclosed herein relate to a
process of isolating 1,4-BDO from a fermentation broth that
includes removing a portion of solids by disc stack centrifugation
to provide a liquid fraction, removing a further portion of solids
from the liquid fraction by ultrafiltration, removing a portion of
salts from the liquid fraction by evaporative crystallization,
removing a further portion of salts from the liquid fraction by ion
exchange, and distilling 1,4-BDO.
[0011] In still other aspects, embodiments disclosed herein relate
to a process of isolating 1,4-BDO from a fermentation broth that
includes removing a portion of solids by disc stack centrifugation
to provide a liquid fraction, removing a further portion of solids
from the liquid fraction by ultrafiltration, removing a portion of
salts from the liquid fraction by nanofiltration, removing a
further portion of salts from the liquid fraction by ion exchange,
evaporating a portion of water, and distilling 1,4-BDO.
[0012] In yet still other aspects, embodiments disclosed herein
relate to a process for producing 1,4-BDO that includes culturing a
1,4-BDO-producing microorganism in a fermentor for a sufficient
period of time to produce 1,4-BDO. The 1,4-BDO-producing
microorganism includes a microorganism having a 1,4-BDO pathway
including one or more exogenous genes encoding a 1,4-BDO pathway
enzyme and/or one or more gene disruptions. The process further
includes isolating 1,4-BDO according to the described isolation
processes.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1 shows a block diagram of steps in the process of
purifying 1,4-BDO from a fermentation broth.
[0014] FIG. 2 shows a cross-section view of disc-stack
centrifuge.
[0015] FIG. 3 shows a cross-section view of a decanter
centrifuge.
[0016] FIG. 4 shows a diagram of a forced circulation
crystallizer.
[0017] FIG. 5 shows a diagram of a forced circulation crystallizer
with a horizontal heat exchanger and baffles in the active
volume.
[0018] FIG. 6 shows a diagram of a draft tube and baffle
crystallizer.
[0019] FIG. 7 shows a diagram of an induced circulation
crystallizer.
[0020] FIG. 8 shows a diagram of a close-type Oslo
crystallizer.
[0021] FIG. 9 shows a diagram of an open-type Oslo
crystallizer.
[0022] FIG. 10 shows a partial cross-section view of a falling film
evaporator.
[0023] FIG. 11 shows a partial cross-section view of a forced
circulation evaporator.
[0024] FIG. 12 shows a partial cross-section view of a plate
evaporator.
[0025] FIG. 13 shows a diagram of a circulation evaporator.
[0026] FIG. 14 shows a diagram of a fluidized bed evaporator.
[0027] FIG. 15 shows a flow diagram of a complete scheme for the
production and isolation of 1,4-BDO.
[0028] FIG. 16 shows a flow diagram of another complete scheme for
the production and isolation of 1,4-BDO.
DETAILED DESCRIPTION OF THE INVENTION
[0029] Fermentation production of commodity chemicals is a useful
alternative to traditional production using nonrenewable fossil
fuel feedstocks. With the ability to utilize renewable feedstocks
such as recycled biomass and the like, the process can prove more
economical and environmentally sound than fossil fuel based
production. Products generated from fermentation can be useful in
many applications. In specific embodiments, the present invention
provides methods for the production of 1,4-BDO. 1,4-Butanediol
(BDO) is a polymer intermediate and industrial solvent. Downstream,
butanediol can be further transformed; for example, by oxidation to
gamma-butyrolactone, which can be further converted to pyrrolidone
and N-methyl-pyrrolidone, or it can undergo hydrogenolysis to
tetrahydrofuran. These compounds have varied uses as polymer
intermediates, solvents, and additives.
[0030] This invention is directed, in part, to processes for
isolating water miscible compounds having boiling points higher
than water from a fermentation while optionally allowing recycle of
other components of the fermentation broth. The process separates
out cell mass, which can include microbial organisms that have been
engineered with gene insertions, gene disruptions or a combination
of insertions and disruptions to produce compounds in useful yields
from a suitable feedstock.
[0031] The cell-free broth, or liquid fraction, can be further
processed by removal of salts. This can be achieved by several
methods before or after removal of some or substantially all of the
water from the fermentation broth. As described above, salts are
not often recovered for recycle in a fermentation process. Usually
any salt recovery involves a salt form of a desired biosynthetic
product such as lactate, citrate or other carboxylate product or
ammonium salts of amine-containing products, rather than media
salts and the like. The process described herein allows for
recovery of media salts and optional recycle back into
fermentation. The isolation process also involves removal of water,
which can be reintroduced into the fermentation system. In the
final purification, the compound produced by fermentation can be
distilled, or recrystallized if solid, from the remaining liquid
fraction after removal of cells, salts, and water. In the case of a
liquid, the final purification can be accomplished by fractional
distillation, for example.
[0032] In some embodiments, the invention is directed to a process
of isolating a water miscible compound of interest having a boiling
point higher than water from a fermentation broth. The process
includes (1) separating a liquid fraction enriched in the compound
from a solid fraction that includes cells; (2) removing water from
the liquid fraction; (3) removing salts from the liquid fraction,
and (4) purifying the compound of interest by distillation or
recrystallization. Steps (2) and (3) above may be performed in
either order, or together.
[0033] Compounds of interest with boiling points higher than water
that are accessible via fermentation, can have boiling points
10.degree. C., 20.degree. C., 30.degree. C., 40.degree. C.,
50.degree. C., 60.degree. C., 70.degree. C., 80.degree. C.,
90.degree. C., 100.degree. C., 150.degree. C., 200.degree. C. and
300.degree. C. higher than water and more, including all values in
between. Compounds of interest having higher boiling points than
water can include, for example, 1,4-BDO, 1,3-BDO, 2,3-BDO, 1,3-PDO,
1,2-PDO (methyl ethyl glycol), 1,2-ethandiol (ethylene glycol),
gamma-butyrolactone (GBL), 1,5-pentanediol, 1,6-hexanediol.
Furthermore, compounds of interest include those that are water
miscible. In some embodiments, such water miscible compounds can be
recalcitrant to conventional extraction procedures. Additionally,
compounds of interest include those that are neutral. As used
herein, a neutral compound refers to a compound that does not
possess functional groups capable of carrying charge, such as
amines, carboxylic acids, sulfonic acids, boronic acids and the
like. Finally, compounds of interest can be sufficiently small so
as to be permeable to a nanofiltration membrane, as described
further below. Exemplary compound classes include alcohols, diols,
triols, such as glycerin, tetraols, polyols and the like.
[0034] In one specific embodiment, the compound of interest is
1,4-BDO. 1,4-BDO has a boiling point of about 230.degree. C. and is
completely miscible with water. Moreover, there are no solvents
that have been identified that can economically extract 1,4-BDO
from the water. As a neutral molecule, isolation by crystallization
of a salt form is precluded. 1,4-BDO has a molecular weight
sufficiently low to pass through a nanofiltration membrane as
described in Example III below. Furthermore, the solubility of
various fermentation media salts in pure 1,4-BDO is relatively low,
as described in Example VI below.
[0035] In some embodiments, the present invention provides a
process of isolating 1,4-butanediol (1,4-BDO) from a fermentation
broth that includes (1) separating a liquid fraction enriched in
1,4-BDO from a solid fraction that includes cells; (2) removing
water from the liquid fraction; (3) removing salts from the liquid
fraction, and (4) purifying 1,4-BDO.
[0036] One skilled in the art will recognize that given the
guidance of the teachings disclosed herein with respect to the
exemplary compound 1,4-BDO, other water miscible compounds of
interest having boiling points higher than water can be isolated
using the same procedures. For example, the methods disclosed
herein are readily modified to enable the isolation of
1,3-butanediol. Therefore, although many embodiments are
exemplified by 1,4-BDO, it is understood that the methods are
readily adaptable to other water miscible compounds of interest
having boiling points higher than water.
[0037] In some embodiments, the invention is directed to a process
of isolating 1,4-butanediol (1,4-BDO) from a fermentation broth.
The process includes separating a liquid fraction enriched in
1,4-BDO from a solid fraction that includes cells. Water is
evaporated from the liquid fraction before or after separating
salts from the liquid fraction. In some embodiments 1,4-BDO is
separated from salts that have crystallized after water removal as
described further below. The salts have a low solubility in 1,4-BDO
such that the separated 1,4-BDO is about 98% salt-free. In some
embodiments, salts are separated by special filtration methods
and/or ion exchange, or chromatographic methods prior to water
removal as described further below.
[0038] As used herein, "isolating" refers to a process that
includes purification steps to obtain a substantially purified
compound of interest. In particular embodiments, a compound of
interest includes 1,4-BDO. A substantially purified compound
includes those that are at least 98% salt free, in some
embodiments, at least 99% salt free in other embodiments, and at
least 99.5% salt free in still other embodiments. A substantially
purified compound also includes those that are also free of other
impurities in addition to salts such that the compound of interest
is at least 98% pure in some embodiments, at least 99% pure in
other embodiments, and at least 99.5% pure in still further
embodiments.
[0039] As used herein, the term "liquid fraction" refers to a
centrate or supernatant liquid obtained upon removal of solid mass
from the fermentation broth. Solid mass removal includes, some,
substantially all, or all of a solid mass. For example, in
centrifugation, the liquid fraction is the centrate or supernatant
which is separated from the solids. The liquid fraction is also the
portion that is the permeate or supernatant obtained after
filtration through a membrane. The liquid fraction is also the
portion that is the filtrate or supernatant obtained after one or
more filtration methods have been applied.
[0040] As used herein, the term "solid fraction" refers to a
portion of the fermentation broth containing insoluble materials.
Such insoluble materials include, for example, cells, cell debris,
precipitated proteins, fines, and the like. Fines refer to small,
usually amorphous solids. Fines can also be created during
crystallization or during removal of water from the fermentation
broth. Fines can be made up of a compound of interest which can be
dissolved and recrystallized out. Fines can include portions of the
solid fraction that are too small to be captured in a membrane
filtration.
[0041] As used herein, the term "salts," used interchangeably with
media salts and fermentation media salts, refers to the dissolved
ionic compounds used in a fermentation broth. Salts in a
fermentation broth can include, for example, sodium chloride,
potassium chloride, calcium chloride, ammonium chloride, magnesium
sulfate, ammonium sulfate, and buffers such as sodium and/or
potassium and/or ammonium salts of phosphate, citrate, acetate, and
borate.
[0042] As used herein, the term "substantially all" when used in
reference to removal of water or salts refers to the removal of at
least 95% of water or salts. "Substantially all" can also include
at least 96%, 97%, 98%, 99%, or 99.9% removal or any value in
between.
[0043] As used herein, the term "gene disruption" or grammatical
equivalents thereof, is intended to mean a genetic alteration that
renders the encoded gene product inactive. The genetic alteration
can be, for example, deletion of the entire gene, deletion of a
regulatory sequence required for transcription or translation,
deletion of a portion of the gene with results in a truncated gene
product or by any of various mutation strategies that inactivate
the encoded gene product. One particularly useful method of gene
disruption is complete gene deletion because it reduces or
eliminates the occurrence of genetic reversions in the
non-naturally occurring microorganisms of the invention.
[0044] As used herein, the term "microorganism" is intended to mean
a prokaryotic or eukaryotic cell or organism having a microscopic
size. The term is intended to include bacteria of all species and
eukaryotic organisms such as yeast and fungi. The term also
includes cell cultures of any species that can be cultured for the
production of a biochemical.
[0045] As used herein, the term "1,4-BDO-producing microorganism"
is intended to mean a microorganism engineered to biosynthesize
1,4-BDO in useful amounts. The engineered organism can include gene
insertions, which includes plasmid inserts and/or chromosomal
insertions. The engineered organism can also include gene
disruptions to further optimize carbon flux through the desired
pathways for production of 1,4-BDO. 1,4-BDO-producing organisms can
include combination of insertions and deletions.
[0046] "Exogenous" as it is used herein is intended to mean that
the referenced molecule or the referenced activity is introduced
into the host microbial organism. The molecule can be introduced,
for example, by introduction of an encoding nucleic acid into the
host genetic material such as by integration into a host chromosome
or as non-chromosomal genetic material such as a plasmid.
Therefore, the term as it is used in reference to expression of an
encoding nucleic acid refers to introduction of the encoding
nucleic acid in an expressible form into the microbial organism.
When used in reference to a biosynthetic activity, the term refers
to an activity that is introduced into the host reference organism.
The source can be, for example, a homologous or heterologous
encoding nucleic acid that expresses the referenced activity
following introduction into the host microbial organism. Therefore,
the term "endogenous" refers to a referenced molecule or activity
that is present in the host. Similarly, the term when used in
reference to expression of an encoding nucleic acid refers to
expression of an encoding nucleic acid contained within the
microbial organism. The term "heterologous" refers to a molecule or
activity derived from a source other than the referenced species
whereas "homologous" refers to a molecule or activity derived from
the host microbial organism. Accordingly, exogenous expression of
an encoding nucleic acid of the invention can utilize either or
both a heterologous or homologous encoding nucleic acid.
[0047] In some embodiments, the invention provides a process of
purifying a compound of interest from a fermentation broth.
Applicable compounds include those having a boiling point higher
than water and a low salt solubility. Compounds of interest also
include those that are water miscible. An exemplary compound of
interest is 1,4-BDO. The process includes separating a liquid
fraction which contains the product of interest, from a solid
fraction which includes the cells mass. The product of interest can
be any compound having a higher boiling point than water. The cell
mass includes the microbial organisms used in the production of the
compound of interest. The solid fraction also includes cell debris,
fines, proteins, and other insoluble materials from the
fermentation.
[0048] The isolation process also includes removing the salts and
water from the liquid fraction. The order in which they are removed
is inconsequential. In some embodiments, there can be partial
removal of salts, followed by removal of substantially all the
water, and then the remaining salts. In other embodiments, there
can be partial removal of water, followed by removal of
substantially all of the salts, and then the remaining water. In
other embodiments, water can be partially removed prior to
separation of the solid fraction from the fermentation broth. In
still other embodiments, final removal of substantially all the
water can be done as part of the purification steps, for example by
distillation. As disclosed below in Example VI, neat 1,4-BDO does
not appreciably solubilize typical fermentation media salts. Thus,
1,4-BDO can be separated from salts by evaporation of the water
from the liquid fraction. As shown in Example V below, salts begin
to crystallize out when 1,4-BDO concentrations are about 30% by
weight. In some embodiments, 1,4-BDO is a least 98% salt free upon
separation of 1,4-BDO from salts crystallized or precipitated by
water removal. As can be seen from Example VI, closely related
homologues ethanediol and propane diol still appreciably solubilize
fermentation salts. Thus, other methods can be employed to remove
salts even after removal of substantially all the water.
[0049] Eventually when the salts and water have been removed, the
remaining liquid or solid can undergo final purification. When the
product of interest is a liquid, purification can be accomplished
by distillation including by fractional distillation or multiple
distillation, for example. When the product of interest is a solid,
purification can be accomplished by recrystallization.
[0050] The overall process for producing and isolating a compound
of interest and recycling various components of the fermentation
broth are summarized in the block flow diagram of FIG. 1. Step 100
is fermentation utilizing carbon feedstock, such as sucrose, to
produce the compound of interest. Step 110 is the separation of
cells from the fermentation broth providing a liquid fraction, with
Step 115 as an optional recycle of the cells. Step 110 has been
exemplified in Examples I and II in which cells and solids are
separated form fermentation broth by centrifugation and
ultrafiltration. In Step 120, salts are separated from the liquid
fraction, with Step 125 as an optional recycle of the salts. Step
120 has been exemplified in Examples III-V, which describe
nanofiltration (Example III) and ion exchange (Example IV), in
which water is still present in the liquid fraction. Example V
shows the separation of salts through crystallization during water
evaporation. Step 130 is the removal of water via evaporation, with
Step 135 as an optional recycle of the water. Step 130 is
exemplified by Example V, which show the evaporation of water which
facilitates salt separation by precipitation. The order of Steps
120 and 130 are interchangeable as described further below. Finally
in step 140 the compound of interest undergoes final
purification.
[0051] In some embodiments, a process of isolating a compound of
interest, including 1,4-BDO, from a fermentation broth involves
separating a liquid fraction enriched in the compound of interest
from a solid fraction that includes cells. In separating a liquid
fraction enriched in the compound of interest, any amount of the
fermentation broth can be processed including 1%, 5%, 10%, 20%,
30%, 40%, 50%, 60%, 70%, 80%, 90%, including up to the entirety of
the volume of the fermentation broth and all values in between, and
further including volumes less than 1% of the total volume of the
fermentation broth. One skilled in the art will recognize that the
amount of fermentation broth processed can depend on the type of
fermentation process, such as batch, fed batch, or continuous, as
detailed below. Separation of solids which includes cells and other
solid byproducts and impurities from the fermentation broth can be
accomplished by centrifugation, filtration, or a combination of
these methods.
[0052] In some embodiments, centrifugation can be used to provide a
liquid fraction comprising the compound of interest, such as
1,4-BDO, substantially free of solids including the cell mass.
Depending on the centrifuge configuration and size, operating
speeds can vary between 500 to 12,000 rpm which produce a
centrifugal force of up to 15,000 times the force of gravity. Many
centrifuge configurations for removal of cells and solids from a
fermentation broth are known in the art. One such configuration,
for example, is the disc-stack centrifuge 200 shown in FIG. 2.
[0053] Separation in a disc-stack centrifuge takes place inside a
rotating bowl 210. The feed is introduced to the rotating bowl from
the top via a stationary inlet pipe 220, and is accelerated in the
distributor 230, before entering the disc stack 240. The
distributor is designed accelerate the feed liquid.
[0054] The separation of liquid-solids or liquid-liquid-solids
takes place between the discs. In a two phase system, such as with
immiscible oil and water phase, the oil phase moving through the
disc stack to the centre and can be discharged through pipes 250
and sprayed out into a collecting frame. The water and solids
separated from the oil move to the periphery, the water is led via
channels in the top disc 260 to the paring chamber, where it is
pumped out of the rotor with means of a built-in paring disc
270.
[0055] The solids are collected in the periphery, from where it can
be discharged intermittently via a centrifuge cyclone. The solids
discharge can be achieved by a hydraulic system which at preset
suitable intervals forces the sliding bowl bottom 280 to drop down
opening the solids port at the bowl periphery.
[0056] A disc stack centrifuge separates solids and one or two
liquid phases from each other, typically in a continuous process.
The denser solids are forced outwards by centrifugal forces while
the less dense liquid phases form inner concentric layers. By
inserting special plates (disc stack) separation efficiency is
increased. The solids can be removed manually, intermittently or
continuously. In accordance with some embodiments, the cell mass
can be introduced back into the fermentation. In a typical
disc-stack centrifuge apparatus, the liquid phase overflows in an
outlet area on top of a bowl into a separate chamber.
[0057] During operation of a disc-stack centrifuge, feed is
introduced at the axis of the bowl, accelerated to speed, often by
a radial vane assembly, and flows through a stack of closely spaced
conical disks. Disk spacing is often between 0.5 to 3 mm in order
to reduce the distance needed for separating settling particles
from the fluid. The disc angle is often between 40 and 50 degrees
to facilitate solids transport down the disk surface into the
solids holding space.
[0058] The separating mechanism is based on the settling of solids
under the influence of centrifugal force against the underside of
the disks and slide down the disk into the solids hold space.
Concurrently the clarified fluid moves up the channel between the
disks and leaves the centrifuge via a centripetal pump. The settled
solids are discharged either continuously though nozzles or
intermittently through ports at the bowl periphery.
[0059] The disc-stack centrifuge can be used at low concentration
and particle size of cells in a fermentation broth. A disc-stack
centrifuge can be employed when the cell and other solid mass
includes as little as about 0.2% to about 3% by weight of the
fermentation broth. The disc-stack centrifuge can also be used when
the cell and other solid mass is less than about 0.2% by weight,
for example, 0.01%, 0.05%, and 0.1% by weight, including all values
in between. The disc-stack centrifuge can also be used when the
cell and other solid mass is more than 3% by weight, for example,
4%, 5%, 6%, 7%, 8%, 9%, 10%, and 15% by weight, including all
values in between. When the combined cell mass and other solids is
higher than about 3% to about 15% by weight other centrifugation
configurations can be used, such as a decanter centrifuge.
[0060] Cells and other solid particles that are soft, plastic, and
not abrasive, ranging from about 0.5 microns to about 500 microns
are generally well-suited for disc-centrifugation. For particulate
matter less than about 0.5 microns, ultrafiltration is useful.
Likewise, above about 500 microns, a decanter-type centrifuge can
be useful. The size of a typical prokaryotic cell that can be
cultured to produce a compound of interest, including 1,4-BDO, can
range in size from about 0.5 microns to about 10 microns, making
disc-stack centrifugation a well-suited method.
[0061] Following batch, or during fed-batch or continuous
fermentation, cells and insoluble solids can be removed from the
fermentation broth by a disc-stack centrifuge. Outputs from a
disc-stack centrifuge are a clarified (cell-free) centrate and an
underflow stream containing about 5% to about 50% solids. The
underflow solids stream from the disc stack centrifuge can contain
a significant amount of the product of interest which can be
recovered. One way to recover additional compound of interest from
the solids is to include further centrifugation steps. In addition
to providing greater recovery of the compound of interest, multiple
centrifugation also serves to further concentrate the cells and
solids. The concentrated cells can be recycled back to the
fermentation. Cell recycle is particularly useful when valuable
engineered organisms are being used.
[0062] In some embodiments, a decanter centrifuge can be employed
to separate out the cells and solids. Good performance with a
decanter centrifuge is normally realized with solids having
particle sizes with a lower limit approaching about 10 microns,
although smaller particles can be processed depending on their
settling speed as described further below. This centrifuge
configuration can be used when the cells of a culture are at the
larger size range of a typical prokaryotic organism. One skilled in
the art will appreciate that eukaryotic cells are often much larger
than prokaryotic cells, with an average eukaryotic cell ranging in
size from about 10 microns to about 100 microns or larger. Although
a disc-stack centrifuge can operate well in this size range, a
decanter centrifuge is useful because it is able to handle larger
amounts of solids. Thus, when the cell mass plus other solids is
more than about 3 to about 50% of the mass by weight, a decanter
centrifuge can be used. This concentration applies to the underflow
of the disc stack centrifuge described above, making a decanter
centrifuge a well suited method to further concentrate the cell
mass and recover additional product.
[0063] The decanter, or solid bowl, centrifuge operates on the
principle of sedimentation. Exemplary apparatus are described in
U.S. Pat. Nos. 4,228,949 and 4,240,578, which are incorporated
herein by reference in their entirety. In such an apparatus 300, as
shown in FIG. 3, the central part of the machine is a rotating drum
310, which contains an independently rotating screw, 320. The
fermentation broth or solids-containing feed, such as the underflow
from the disc stack centrifuge, is fed via the inlet pipe 330 to
the mixing chamber 340 in the core of the first part of the screw.
The broth then passes through ports in the mixing chamber out
towards the outer walls of the drum. The dewatered broth is
transported out through the machine by the screw. The centrate 350,
or supernatant, is decanted from the inner surface of the pond
through centrate pipes. The water level in the drum can be adjusted
in accordance with the characteristics of the material to be
processed.
[0064] The drum and the screw rotate independently of one another
at speeds up to about 3,600 rpm, depending on the type and size of
machine. The dewatering principles used are known in the art as the
"concurrent" or "counter-current" method. The concurrent method
permits very low differential speeds. The differential speed is the
difference between the speed of the drum and the speed of the
screw. Low differential speeds mean longer residence times in the
centrifuge, which result in drier sludge and considerably less
wear. The counter-current principle can be more suitable for a feed
that is easy to dewater and when a high capacity is desired.
[0065] Solids can be separated in solid bowl centrifuges provided
their sedimentation speed in the liquid phase portion of the feed
is sufficient. Factors that influence sedimentation speed include,
for example, particle size, shape, differences in density between
the cells/solids and the fermentation broth liquid phase, and
viscosity. The geometry of the bowl, especially the relation
between the length and diameter, are adaptable to suit the
particular conditions. In some embodiments, good results can be
obtained at length diameter ratio ranging from about 2:1 to about
3:1.
[0066] In operation, separation takes place in a horizontal conical
cylindrical bowl equipped with a screw conveyor. The fermentation
broth is fed into the bowl through a stationery inlet tube and
accelerated by an inlet distributor. Centrifugal force provides the
means for sedimentation of the solids on the wall of bowl. A
conveyor, rotating in the same direction as bowl with differential
speed, conveys the solids to the conical end. The solids are then
lifted clear of the liquid phase and centrifugally dewatered before
being discharged into a collecting channel. The remaining liquid
phase then flows into a housing through an opening in cylindrical
end of the bowl.
[0067] As described above, the cells and solids can be separated by
multiple centrifugation to increase the isolated yield of the
compound of interest. Multiple centrifugation can include
centrifugation twice, three times, four times, and five times, for
example. Intermediate underflow streams can be diluted with water
to further increase recovery of the liquid product. Any combination
of centrifugation configurations can also be used to perform
multiple centrifugations, such as combinations of the disc-stack
and decanter centrifugations described above. Further solids that
are not separable by centrifugation can be removed through a
filtration process, such as ultrafiltration.
[0068] Ultrafiltration is a selective separation process through a
membrane using pressures up to about 145 psi (10 bar). Useful
configurations include cross-flow filtration using spiral-wound,
hollow fiber, or flat sheet (cartridge) ultrafiltration elements.
These elements consist of polymeric or ceramic membranes with a
molecular weight cut-off of less than about 200,000 Daltons, for
example Hydranautics 5K PES membrane as used in Example I below.
Ceramic ultrafiltration membranes are also useful since they have
long operating lifetimes of up to or over 10 years. Ceramics have
the disadvantage of being much more expensive than polymeric
membranes. Ultrafiltration concentrates suspended solids and
solutes of molecular weight greater than about 1,000 Daltons.
Ultrafiltration includes filtering through a membrane having
nominal molecular weight cut-offs (MWCO) from about 1,000 Daltons
to about 200,000 Daltons (pore sizes of about 0.005 to 0.1
microns). The term molecular weight cut-off is used to define the
size of protein that will be approximately 90% retained by the
membrane. Using ultrafiltration the permeate liquid will contain
low-molecular-weight organic solutes, such as 1,4-BDO, media salts,
and water. The captured solids can include, for example, residual
cell debris, DNA, and proteins.
[0069] In addition to the use ultrafiltration downstream of
centrifugation, ultrafiltration can also be used downstream of
microfiltration. Microfiltration provides an alternate means to
centrifugation for separating cells. Microfiltration usually
involves a low-pressure cross-flow membrane process for separating
colloidal and suspended particles in the range of about 0.05-10
microns. Microfiltration includes filtering through a membrane
having pore sizes from about 0.05 microns to about 5.0 microns.
Polymeric, ceramic, or steel microfiltration membranes can be used
to separate cells. Ceramic or steel microfiltration membranes have
long operating lifetimes including up to or over 10 years.
Microfiltration can be used in the clarification of fermentation
broth. Unlike ultrafiltration, microfiltration will generally not
capture residual cell debris, DNA, and proteins. However, it is
useful to use a series of filtration steps with gradually
decreasing pore size in order to avoid fouling of the filter
membranes. This is useful for optimizing reuse of the filter
membrane. In some embodiments, a single ultrafiltration step can be
used to remove both cell mass (in place of centrifugation or
microfiltration) and residual cell debris, DNA, proteins, etc.
Ceramic ultrafiltration elements are useful for this application
due to their ability to tolerate the frequent cleaning cycles used
in this mode of operation.
[0070] In some embodiments, a further filtration method called
nanofiltration can be used to separate out certain salts. This
process step can allow the recovery of certain media salts without
prior evaporation of water, for example. Nanofiltration can
separate salts, remove color, and provide desalination. In
nanofiltration, the permeate liquid generally contains monovalent
ions and low-molecular-weight organic compounds as exemplified by
1,4-BDO. Nanofiltration includes filtering through a membrane
having nominal molecular weight cut-offs (MWCO) from about 100
Daltons to about 2,000 Daltons (pore sizes of about 0.0005 to 0.005
microns). One method for nanofiltration is cross-flow filtration
using a spiral-wound element. There are several nanofiltration
membranes available, for example the thin film composite
nanofiltration membrane GE DK used in Example III below. The mass
transfer mechanism in nanofiltration is diffusion. The
nanofiltration membrane allows the partial diffusion of certain
ionic solutes (such as sodium and chloride), predominantly
monovalent ions, as well as water. Larger ionic species, including
divalent and multivalent ions, and more complex molecules are
substantially retained.
[0071] Since monovalent ions are partially diffusing through the
nanofiltration membrane along with the water, the osmotic pressure
difference between the solutions on each side of the membrane is
not as great and this typically results in somewhat lower operating
pressure with nanofiltration compared with, for example, reverse
osmosis.
[0072] Nanofiltration not only removes a portion of the inorganic
salts but can also remove salts of organic acids. The removal of
organic acid byproducts can be important in the isolation process
because such acids can catalyze or serve as a reactant in
undesirable side reactions with a product of interest. In the
context of specific embodiments related to the isolation of
1,4-BDO, for example, the removal of organic acids is particularly
useful because it can prevent reactions such as esterification of
the hydroxyl groups during the elevated temperatures of any
downstream evaporation or distillation steps. These ester
byproducts typically have higher boiling points than BDO resulting
in yield losses to the heavies stream in distillation.
[0073] Nanofiltration can also separate the glucose or sucrose
substrate from the product of interest, preventing degradation
reactions during evaporation and distillation. These degradation
reactions can produce coloration of the compound of interest. The
salt and substrate rich nanofiltration retentate can be better
suited for recycle to fermentation compared to a recovered salt
stream from evaporative crystallization. For example, the use of
filtration methods in lieu of methods involving application of heat
can result in fewer degradation products. Such degradation products
can be toxic to the fermentation organism.
[0074] Both nanofiltration and ion exchange can remove color
forming compounds and UV absorbing compounds. This can be useful in
the context of some compounds of interest. For example, color
removal is useful in the production of polymer grade 1,4-BDO.
[0075] Multiple filtration membranes can be used serially with
gradually increasing refinement of the size of the solids that are
retained. This can be useful to reduce fouling of membranes and aid
in recovering individual components of the fermentation broth for
recycle. For example, a series of filtrations can utilize
microfiltration, followed by ultrafiltration, followed by
nanofiltration. Thus, microfiltration aids in recovery of cell
mass, ultrafiltration removes large components such as cell debris,
DNA, and proteins, and nanofiltration aids in recovery of
salts.
[0076] Those skilled in the art will recognize that any of the
various filtration types can be integrated within the context of a
variety of fermentation bioreactor configurations given the
teachings and guidance provide herein. In some embodiments the
filtration occurs external to the bioreactor. In this mode, any
amount of the fermentation broth can be removed from the bioreactor
and filtered separately. Filtration can be aided by use of vacuum
methods, or the use of positive pressure. In some embodiments, cell
filtration can be accomplished by means of a filtration element
internal to the bioreactor. Such configurations include those found
in membrane cell-recycle bioreactors (MCRBs). Chang et al. U.S.
Pat. No. 6,596,521 have described a two-stage cell-recycle
continuous reactor.
[0077] In some embodiments, the cells can be separated and recycled
into the fermentation mixture by means of an acoustic cell settler
as described by Yang et al. (Biotechnol. Bioprocess. Eng.,
7:357-361(2002)). Acoustic cell settling utilizes ultrasound to
concentrate the suspension of cells in a fermentation broth. This
method allows for facile return of the cells to the bioreactor and
avoids the issue of membrane fouling that sometimes complicates
filtration-type cell recycle systems.
[0078] With respect to isolation of salts prior to water
evaporation, other methods can be used alone, or in combination
with the above exemplary filtration processes. Such other methods
include, for example, ion exchange. For example, Gong et al.
(Desalination 191:1-3, 193-199 (2006)) have described the effects
of transport properties of ion-exchange membranes on desalination
of 1,3-propanediol fermentation broth by electrodialysis.
[0079] Ion exchange elements can take the form of resin beads as
well as membranes. Frequently, the resins are cast in the form of
porous beads. The resins can be cross-linked polymers having active
groups in the form of electrically charged sites. At these sites,
ions of opposite charge are attracted but may be replaced by other
ions depending on their relative concentrations and affinities for
the sites. Ion exchangers can be cationic or anionic, for example.
Factors that determine the efficiency of a given ion exchange resin
include the favorability for a given ion, and the number of active
sites available. To maximize the active sites, large surface areas
are generally useful. Thus, small particles are useful because of
their large surface area.
[0080] The resin polymer can include cross-linking on the order of
about 0.5 to about 15 percent, for example. Temperature and pH also
affect the efficiency of ion exchange. For example, pH can affect
the number of ions available for exchange, and temperature affects
the kinetics of the process. In some embodiments, salt removal by
ion exchange includes removal of organic acids and salts of organic
acids. The anionic form of organic acids can bind to an anion
exchange active site. In some embodiments, the pH for binding an
organic acid is below the pKa for that acid. The pKa of lactic
acid, for example, is about 3.1. An effective method for removing
salts of organic acids is cation exchange followed by anion
exchange. The cation resin first removes the organic acid
counter-ion (calcium, sodium, ammonium, and the like), lowering the
pH of the solution. The anion resin then binds the free acid.
[0081] A useful aspect of ion exchange is the facility with which
the resin can be regenerated. The resin can be flushed free of the
exchanged ions and contacted with a solution of desirable ions to
replace them. With regeneration, the same resin beads can be used
over and over again, and the isolated ions can be concentrated in a
waste effluent. As with the many filtration methods, serial ion
exchange can be performed, as exemplified in Example IV. Thus, a
feed can be passed through both any number of anionic and cationic
exchangers, or mixed-bed exchangers, and in any order.
[0082] In some embodiments, water removal via evaporation is used
to facilitate salt recovery. In some embodiments, the salts have
been removed prior to water removal. In either case, evaporated
water can be recycled as makeup water to the fermentation,
minimizing the overall water requirements for the process. In the
case where the salts have not been removed, their solubility in the
1,4-BDO enriched liquid phase is sufficiently low that they can
crystallize after water removal. In some embodiments the salts have
a sufficiently low solubility in 1,4-BDO that the separated 1,4-BDO
is about 98% salt-free.
[0083] An evaporative crystallizer can be used to generate
precipitated salts which can be removed by centrifugation,
filtration or other mechanical means. In the context of 1,4-BDO
isolation, an evaporative crystallizer serves to remove water from
the fermentation broth creating a liquid phase that has removed
enough water to cause supersaturation of the fermentation media
salts and subsequent crystallization in the remaining liquid phase
or mother liquor. As demonstrated in Example V below,
crystallization of salts begins at a 1,4-BDO concentration of about
30% by weight.
[0084] The mother liquor refers to the bulk solvent in a
crystallization. Frequently, the mother liquor is a combination of
solvents with different capacity to solublize or dissolve various
solutes. In the context of the purification of 1,4-BDO from a
fermentation broth, for example, the mother liquor includes the
liquid fraction obtained after removing cells and other solids from
the fermentation broth. In the context of isolating a compound of
interest from a fermentation broth, the primary solute includes the
fermentation media salts and organic acids.
[0085] Supersaturation in crystallization refers to a condition in
which a solute is more concentrated in a bulk solvent than is
normally possible under given conditions of temperature and
pressure. The bulk solvent of the fermentation broth is water
containing relatively smaller amounts of 1,4-BDO, for example, and
dissolved salts and other media.
[0086] An exemplary evaporative crystallizer is the forced
circulation (FC) crystallizer as shown in FIGS. 5 and 6. An FC
crystallizer has been described, for example, in U.S. Pat. No.
3,976,430 which is incorporated by reference herein in its
entirety. The FC crystallizer evaporates water resulting in an
increased supersaturation of the salts in the compound-enriched
(such as 1,4-BDO) liquid fraction thus causing the salts to
crystallize. The FC crystallizer is useful for achieving high
evaporation rates. The FC crystallizer consists of four basic
components: a crystallizer vessel with a conical bottom portion, a
circulating pump, a heat exchanger, and vacuum equipment which
handles the vapors generated in the crystallizer. Slurry from the
crystallizer vessel is circulated through the heat exchanger, and
returned to the crystallizer vessel again, where supersaturation is
relieved by deposition of salts on the crystals present in the
slurry. The evaporated water is conducted to the vacuum system,
where it is condensed and recycled to the fermentation broth as
desired. Although in some embodiments, there is a low vacuum, it is
also possible to use the FC crystallizer at about atmospheric
pressure as well. In some embodiments, the FC crystallizer utilizes
adiabatic evaporative cooling to generate salt supersaturation. In
such embodiments, the FC crystallizer need not be equipped with a
heat exchanger.
[0087] In some embodiments, the FC crystallizer can be further
equipped with internal baffles, as shown in FIG. 6, to handle
overflow of the liquid phase and to reduce fines which can inhibit
crystal growth. The salts generated in the FC crystallizer can also
be size selected with the aid of an optional elutriation leg. This
portion of the FC crystallizer appears at the bottom of the conical
section of the crystallizer vessel. Size selection is achieved by
providing a flow of fermentation fluid up the leg allowing only
particles with a particular settling rate to move against this
flow. The settling speed is related to the size and shape of the
crystals as well as fluid viscosity. In further embodiments, the FC
crystallizer can also be equipped with an internal scrubber to
reduce product losses. This can assist in the recovery of volatile
products.
[0088] The turbulence or draft tube and baffle "DTB" crystallizer,
shown in FIG. 7, provides two discharge streams, one of a slurry
that contains crystals, and another that is the liquid phase with a
small amount of fines. The configuration of the DTB crystallizer is
such that it promotes crystal growth, and can generate crystals of
a larger average size than those obtained with the FC crystallizer.
In some embodiments, the DTB crystallizer operates under vacuum, or
at slight superatmospheric pressure. In some embodiments, the DTB
crystallizer uses vacuum for cooling.
[0089] In some embodiments, a DTB crystallizer operates at a low
supersaturation. One skilled in the art will appreciate that large
crystals can be obtained under this regime. The system can be
optionally configured to dissolve fines to further increase crystal
size. When the DTB crystallizer is used in fermentation media salt
recovery, crystal size is not necessarily a priority.
[0090] The DTB crystallizer has been studied widely in
crystallization, and can be modeled with accuracy. Its distinct
zones of growth and clarified liquid phase facilitate defining
kinetic parameters, and thus, the growth and nucleation rate can be
readily calculated. These features make the DTB crystallizer
suitable to mathematical description, and thus, subject to good
operating control. The DTB crystallizer is an example of a mixed
suspension mixed product removal (MSMPR) design, like the FC
crystallizer.
[0091] The DTB crystallizer includes a baffled area, serving as a
settling zone, which is peripheral to the active volume. This zone
is used to further process the liquid phase and fines. In some
embodiments, the baffled area is not present, as can be the case
where further processing of fines is less important. Such a
configuration is known in the art as a draft-tube crystallizer. A
DTB crystallizer can be equipped with an agitator, usually at the
bottom of the apparatus in the vicinity of the entry of the feed
solution. Like the FC crystallizer, the DTB crystallizer is
optionally equipped with an elutriation leg. In some embodiments,
an optional external heating loop can be used to increase
evaporation rates.
[0092] Yet another crystallizer configuration is the induced
circulation crystallizer as shown in FIG. 8. This configuration
provides additional agitation means for the active volume. The
apparatus is similar to the DTB crystallizer with respect to the
use of a draft tube. Unlike the DTB apparatus, there is no internal
agitator. Instead, an inducer in the conical portion of the vessel
introduces heated solution from a recirculation pump. As with other
crystallization apparatus configurations, the induced circulation
crystallizer is optionally equipped with an elutriation leg.
Baffles can also be optionally employed with this type of
crystallizer.
[0093] In still further embodiments, the crystallizer can be an
Oslo-type crystallizer, as shown in FIGS. 9 and 10. This type of
crystallizer is also referred to as "growth-", "fluid-bed-", or
"Krystal-" type crystallizer. The Oslo crystallizer allows the
growth of crystals in a fluidized bed, which is not subject to
mechanical circulation. A crystal in an Oslo unit will grow to a
size proportional to its residence time in the fluid bed. The
result is that an Oslo crystallizer can grow crystals larger than
most other crystallizer types. The slurry can be removed from the
crystallizer's fluidized bed and sent to, for example, a
centrifugation section. Clear liquid phase containing 1,4-BDO can
be purged from the crystallizer's clarification zone.
[0094] The classifying crystallization chamber is the lower part of
the unit. The upper part is the liquor-vapor separation area where
supersaturation is developed by the removal of water. The slightly
supersaturated liquid phase flows down through a central pipe and
the supersaturation is relieved by contact with the fluidized bed
of crystals. The desupersaturation occurs progressively as the
circulating liquid phase moves upwards through the classifying bed
before being collected in the top part of the chamber. The
remaining liquid leaves via a circulating pipe and after addition
of the fresh feed, it passes through the heat exchanger where heat
make-up is provided. It is then recycled to the upper part.
[0095] In some embodiments, the Oslo type crystallizer can also be
optionally equipped with baffles, an elutriation leg, and scrubber
as described above. Since the growing crystals are not in contact
with any agitation device, the amount of fines to be destroyed is
generally lower. The Oslo type crystallizer allows long cycles of
production between periods for crystal removal.
[0096] The Oslo-type crystallizer is useful for the
separation-crystallization of several chemical species as would be
found in fermentation media salts. In one embodiment, the Oslo type
crystallization unit is of the "closed" type, as shown in FIG. 9.
In other embodiments the Oslo-type crystallizer is the "open" type
as shown in FIG. 10. The latter configuration is useful when large
settling areas are needed, for example.
[0097] Many of the foregoing evaporative crystallization apparatus
allow for controlled crystal growth. In the recovery of
fermentation media salts from the liquid portion after cell
removal, the exact crystal morphology, size, and the like are
generally inconsequential. Indeed, recovery of amorphous media
salts can be sufficient in the purification of any compound of
interest, including 1,4-BDO. Thus, in some embodiments, other
evaporation methods can be utilized that do not control crystal
growth per se.
[0098] When salts are removed by nanofiltration and/or ion
exchange, a reverse osmosis (RO) membrane filtration can be used to
remove a portion of the water prior to evaporation. Water permeates
the RO membrane while 1,4-BDO is retained. In some embodiments, an
RO membrane can concentrate a product, such as 1,4-BDO to about
20%. One skilled in the art will recognize that the osmotic
pressure from the product 1,4-BDO increases to a point where
further concentration using an RO membrane is no longer viable.
Nonetheless, the use of an RO membrane is a useful low energy input
method for concentrating the product of interest prior to the more
energy intensive water evaporation process. Thus, on large scale,
employing a RO membrane is particularly useful.
[0099] In some embodiments, substantially all of the salts are
removed prior to removal of water. In other embodiments,
substantially all of the salts are removed after removal of a
portion of water. The portion of water removed can be any amount
including 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%,
80%, and all values in between. In some embodiments, salts are
removed after removal of substantially all of the water.
Substantially all the water includes 95%, 96%, 97%, 98%, 99%, 99.9%
and all values in between and including all the water.
[0100] There are many types and configurations of evaporators
available for water removal. One consideration for designing an
evaporation system is minimizing energy requirements. Evaporation
configurations such as multiple effects or mechanical vapor
recompression allow for reduced energy consumption. In some
embodiments, removing water is accomplished by evaporation with an
evaporator system which includes one or more effects. In some
embodiments, a double- or triple-effect evaporator system can be
used to separate water from a product of interest, such as 1,4-BDO.
Any number of multiple-effect evaporator systems can be used in the
removal of water. These apparatus can also be applied to any
fermentation product that having a boiling point higher than water.
A triple effect evaporator, or other evaporative apparatus
configuration, can include dedicated effects that are evaporative
crystallizers for salt recovery, for example the final effect of a
triple effect configuration.
[0101] An evaporator is a heat exchanger in which a liquid is
boiled to give a vapor that is also a low pressure steam generator.
This steam can be used for further heating in another evaporator
called another "effect." Thus, for example, two evaporators can be
connected so that the vapor line from one is connected to the steam
chest of the other providing a two, or double-effect evaporator.
This configuration can be propagated to a third evaporator to
create a triple-effect evaporator, for example.
[0102] Evaporators can therefore be classified by the number of
effects. In a single-effect evaporator, steam provides energy for
vaporization and the vapor product is condensed and removed from
the system. In a double-effect evaporator, the vapor product off
the first effect is used to provide energy for a second
vaporization unit. The cascading of effects can continue for any
number of stages. Multiple-effect evaporators can remove large
amounts of solvent more efficiently relative to a single effect
evaporator.
[0103] In a multiple effect arrangement, the latent heat of the
vapor product off of an effect is used to heat the following
effect. Effects are numbered beginning with the one heated by
steam, Effect I. The first effect operates under the highest
pressure. Vapor from Effect I is used to heat Effect II, which
consequently operates at lower pressure. This continues through
each addition effect, so that pressure drops through the sequence
and the hot vapor will travel from one effect to the next.
[0104] In some embodiments, all effects in an evaporator can be
physically similar in size, construction, and heat transfer area.
Unless thermal losses are significant, they can also have the same
capacity as well. Evaporator trains, the serially connected
effects, can receive feed in several different ways. Forward Feed
arrangements follow the pattern I, II, and III. These use a single
feed pump. In this configuration the feed is raised to the highest
operating temperature as used in Effect I. The lowest operating
temperature is in the final effect, where the product is also most
concentrated. Therefore, this configuration is useful for products
that are heat sensitive or to reduce side reactions.
[0105] In other embodiments, Backward Feed arrangements, III, II, I
can be used. In such a configuration multiple pumps are used to
work against the pressure drop of the system, however, since the
feed is gradually heated they can be more efficient than a forward
feed configuration. This arrangement also reduces the viscosity
differences through the system and is thus useful for viscous
fermentation broths. In some embodiments, Mixed Feed arrangements
can be utilized, with the feed entering in the middle of the
system, or effects II, III, and I. The final evaporation is
performed at the highest temperature. Additionally, fewer pumps are
required than in a backward feed arrangement. In still further
embodiments, a Parallel Feed system is used to split the feed
stream and feed a portion to each effect. This configuration is
common in crystallizing evaporators where the product is expected
to be a slurry.
[0106] There are numerous evaporator designs. Any combination of
designs can be used as an effect as described above. One evaporator
design is the falling film evaporator. This apparatus includes a
vertical shell-and-tube heat exchanger, with a laterally or
concentrically arranged centrifugal separator as shown in FIG.
11.
[0107] The liquid to be evaporated is evenly distributed on the
inner surface of a tube. The liquid flows downwards forming a thin
film, from which evaporation takes place because of the heat
applied by the steam. The steam condenses and flows downwards on
the outer surface of the tube. A number of tubes are built together
side by side. At each end the tubes are fixed to tube plates, and
finally the tube bundle is enclosed by a jacket.
[0108] The steam is introduced through the jacket. The space
between the tubes forms the heating section. The inner side of the
tubes is called the boiling section. Together they form the
calandria. The concentrated liquid and the vapor leave the
calandria at the bottom part, from where the main proportion of the
concentrated liquid is discharged. The remaining part enters the
subsequent separator tangentially together with the vapor. The
separated concentrate is discharged, usually be means of the same
pump as for the major part of the concentrate from the calandria,
and the vapor leaves the separator from the top. The heating steam,
which condenses on the outer surface of the tubes, is collected as
condensate at the bottom part of the heating section, from where it
is discharged.
[0109] Falling film evaporators can be operated with very low
temperature differences between the heating media and the boiling
liquid, and they also have very short product contact times,
typically just a few seconds per pass. These characteristics make
the falling film evaporator particularly suitable for
heat-sensitive products. Operation of falling film evaporators with
small temperature differences facilitates their use in multiple
effect configurations or in conjunction with mechanical vapor
compression systems.
[0110] Sufficient wetting of the heating surface in tubes of the
calandria helps avoid dry patches and incrustations which can clog
the tubes. In some embodiments, the wetting rate can be increased
by extending or dividing the evaporator effects. Falling film
evaporators are highly responsive to alterations of parameters such
as energy supply, vacuum, feed rate, and concentrations, for
example. In some embodiments, a single, double, triple, or other
multiple-effect falling film evaporator configuration can utilize
fermentation feed that has been filtered through a nanofiltration
process as detailed above. Reducing the salts prior to water
evaporation can further help prevent incrustation in the tubes of
the calandria.
[0111] In some embodiments, the falling film evaporator is a short
path evaporator. In operation the liquid fraction is evenly
distributed over the heating tubes of the calandria by means of a
distribution system. The liquid fraction flows down in a thin film
on the inside walls in a manner similar to the conventional falling
film evaporator. The vapors formed in the in the calandria tubes
are condensed as a distillate on external walls of condensate tubes
and then flows downward. Water distillate and the enriched liquid
fraction are separately discharged from the lower part of the
evaporator.
[0112] Another evaporator configuration is the forced circulation
evaporator. In this design a flash vessel or separator is disposed
above a calandria and circulation pump as shown in FIG. 12. In
operation, the liquid fraction is circulated through the calandria
by means of a circulation pump. The liquid is superheated within
the calandria at an elevated pressure higher than the normal
boiling pressure. Upon entering the separator, the pressure is
rapidly reduced resulting in flashing or rapid boiling of the
liquid. The flow velocity, controlled by the circulation pump, and
temperatures can be used to control the water removal process. This
configuration is useful for avoiding fouling of the calandria
tubes.
[0113] In some embodiments, multiple forced circulation evaporator
effects can be used as described above. For example, in addition to
a single effect forced circulation evaporator, double, triple, and
multiple effect forced circulation evaporators can be used in the
separation of water from the liquid fraction of the fermentation
liquid. In some embodiments, one or more forced circulation
evaporators can be used in conjunction with one or more falling
film evaporators.
[0114] In still further embodiments, the evaporator can be a plate
evaporator, as shown in FIG. 13. This evaporator uses a plate heat
exchanger and one or more separators. A plate-and-frame
configuration uses plates with alternating channels to carry
heating media and the liquid fraction of the fermentation broth. In
operation, the liquid phase and heating media are passed through
their respective channels in counterflow. Defined plate distances
and shapes generate turbulence resulting in efficient heat
transfer. The heat transfer to the channels with the liquid
fraction causes water to boil. The vapor thus formed drives the
residual liquid as a rising film into a vapor duct of the plate
assembly. Residual liquid and vapors are separated in the
downstream centrifugal separator. The wide inlet duct and the
upward movement assist in good distribution over the cross-section
of the heat exchanger. A plate evaporator can be usefully operated
with a pre-filtration through a nanofiltration membrane to avoid
fouling. Thus, similar considerations as the falling film
evaporator with respect to incrustation are warranted.
[0115] In some embodiments, multiple-effect plate evaporation can
be utilized in much the same manner as described above for falling
film and forced circulation evaporators. When used in multiple
effect configurations, one skilled in the art will recognize the
benefit of using a forced circulation evaporator and/or a
nanofiltration step prior to introduction of the liquid fraction to
a plate evaporator. Thus, a separation scheme can include, for
example, nanofiltration, followed by a multiple-effect evaporation
configuration of one or more forced circulation evaporators,
followed by one or more of a plate and/or falling film evaporator.
In still further embodiments, any of the evaporative crystallizers
described above can also be used in conjunction with a
multiple-effect configuration.
[0116] In some embodiments, a circulation evaporator can be used to
remove water from the liquid fraction as shown in FIG. 14. The
circulation evaporator utilizes a vertical calandria with short
tube length with a lateral separator disposed at the top of the
heat exchanger. In operation the liquid fraction is supplied at the
bottom of the calandria and rises to the top. During heating in the
tubes of the calandria, the water begins to boil releasing vapor.
The liquid is carried to the top of the calandria entrained by the
upward moving vapors. The liquid is separated from the vapors as it
enters the separator. The liquid flows back into the evaporator via
a circulation pipe to allow continued circulation. The larger the
temperature difference between the heating elements of the
calandria and the separator chamber results in larger degree of
water evaporation from the liquid fraction. When the liquid portion
is sufficiently enriched in 1,4-BDO, the salts will begin to
precipitate from the liquid fraction.
[0117] In some embodiments, the separator of the circulation
evaporator can be partitioned into several separation chambers each
equipped with its own liquid circulation system. This can reduce
the heating surface needed to remove water from the liquid
fraction.
[0118] The fluidized bed evaporator is yet another configuration
that can be used for water removal from the liquid fraction. Such a
system, shown in FIG. 15, is equipped with a vertical fluidized bed
heat exchanger. On the tube side of the heat exchanger are solid
particles such as glass or ceramic beads, or steel wire
particles.
[0119] The fluidized bed evaporator operates in a similar manner to
the forced circulation evaporator. The upward movement of the
liquid entrains the solid particles which provides a scouring or
cleaning action. Together with the liquid fraction they are
transferred through the calandria tubes. At the head of the
calandria, the solid particles are separated from the liquid and
are recycled to the calandria inlet chamber. The superheated fluid
is flashed to boiling temperature in the separator allowing removal
of water through evaporation. The scouring action of the solids in
the tubes of the calandria allow for prolonged operation times and
further retard fouling of the tubes. This can be useful when the
creation of fouling solids limits the use of conventional forced
circulation evaporator systems.
[0120] The rising film evaporator is yet another type of evaporator
useful in the removal of water from the liquid fraction collected
from the fermentation broth. This system configuration has a
top-mounted vapor separator on a vertical shell-and-tube heat
exchanger (calandria). In operation, the liquid fraction at the
bottom of the calandria rises to the top to the vapor separator.
External heating causes the water in the liquid fraction to boil in
the inside walls of the calandria tubes. The upward movement of the
steam causes the liquid fraction to be carried to the top of the
calandria. During ascent though the tube further vapor is formed.
Upon entry into the separator vapors and liquid phases are
separated. The rising film evaporator is particularly useful when
used with viscous liquids and/or when large amounts of fouling
solids are expected.
[0121] The counterflow-trickle evaporator is yet another evaporator
that can be used for water removal from the liquid fraction of the
fermentation broth. This apparatus has a shell-and-tube heat
exchanger (calandria) with the lower part of the calandria larger
than that of a rising film evaporator. Disposed on top of the
calandria, like the rising film evaporator is a separator. In this
evaporator the separator is further equipped with a liquid
distribution system.
[0122] In operation, liquid is provided at the top of the
evaporator like a falling film evaporator. The liquid is
distributed over the evaporator tubes, but vapor flows to the top
in counterflow to the liquid. In some embodiments, the process can
also include a stream of an inert gas, for example, to enhance
entrainment. This gas can be introduced in the lower portion of the
calandria.
[0123] A stirrer evaporator is yet another type of evaporator that
can be used for water removal from the liquid fraction of the
fermentation broth. This apparatus includes an external,
jacket-heated vessel equipped with a stirrer. In operation, the
liquid fraction is placed in the vessel, optionally in batches. The
water is evaporated off by boiling with continuous stirring to a
desired concentration. This apparatus can increase its evaporation
rate by increasing the heating surface by use of optional immersion
heating coils. This type of evaporator is particularly useful when
the fermentation is highly viscous.
[0124] Finally, the spiral tube evaporator is another type of
evaporator that can be used for water removal from the liquid
fraction of the fermentation broth. The design includes a heat
exchanger equipped with spiral heating tubes and a bottom-mounted
centrifugal separator. In operation, the liquid fraction flows a
boiling film from top to bottom in parallel flow to the vapor. The
expanding vapors produce a shear, or pushing effect on the liquid
film. The curvature of the path of flow induces a secondary flow
which interferes with the movement along the tube axis. This
turbulence improves heat transfer and is particularly useful with
viscous liquids. The spiral configuration of the heating tubes
usefully provides a large heating surface area to height ratio
relative to a non-spiral, straight tube design. This apparatus
provides large evaporation ratios allowing single pass
operation.
[0125] As described above, the use of multiple evaporators of any
type described above in double, triple, and multi-effect
configurations can increase the efficiency of evaporation. Other
methods to improve efficiency of operation include, for example,
thermal and mechanical vapor recompression. In some embodiments,
any combination of multiple-effect configurations, thermal
recompression, and mechanical recompression can be used to increase
evaporation efficiency.
[0126] Thermal vapor recompression involves recompressing the vapor
from a boiling chamber (or separator) to a higher pressure. The
saturated steam temperature corresponding to the heating chamber
pressure is higher so that vapor can be reused for heating. This is
accomplished with a steam jet vapor recompressor which operates on
the steam jet pump principle. Briefly, the steam jet principle
utilizes the energy of steam to create vacuum and handle process
gases. Steam under pressure enters a nozzle and produces a high
velocity jet. This jet action creates a vacuum that draws in and
entrains gas. The mixture of steam and gas is discharged at
atmospheric pressure. A quantity of steam, called motive steam, is
used to operate the thermal recompressor. The motive steam is
transferred to the next effect or to a condenser. The energy of the
excess vapor is approximately that of the motive steam quantity
used.
[0127] In multiple-effect evaporators equipped with thermal vapor
recompressors, the heating medium in the first calandria is the
product vapor from one of the associated effects, compressed to a
higher temperature level by means of a steam ejector. The heating
medium in any subsequent effect is the vapor generated in the
previous calandria. Vapor from the final effect is condensed with
incoming product, optionally supplemented by cooling water as
necessary. All recovered water is readily recycled to a
fermentation broth.
[0128] Mechanical recompressors utilize all vapor leaving one
evaporator. The vapor is recompressed to the pressure of the
corresponding heating steam temperature of the evaporator. The
operating principle is similar to a heat pump. The energy of the
vapor condensate can be optionally used to pre-heat further
portions of the liquid fraction of the fermentation broth. The
mechanical recompression is supplied by use of a high pressure fans
or turbocompressors. These fans operate a high velocity and are
suited for large flow rates at vapor compression ratios of about
1:1.2 to about 1:2. Rational speeds can be between about 3,000 to
about 18,000 rpm. In some embodiments, when particularly high
pressures are useful, multiple stage compressors can be used.
[0129] In evaporators with equipped with mechanical vapor
recompressors, the heating medium in the first effect is vapor
developed in the same effect, compressed to a higher temperature by
means of a high-pressure fan. Any excess vapor from the high heat
section is optionally condensed or can be utilized in a high
concentrator.
[0130] As described above there are many possible evaporation types
that can be arranged in various energy efficient configurations
including multiple effect, thermal vapor recompression, mechanical
vapor recompression, or combinations of these. Optimal
configurations depend on many factors, including, for example,
whether media salts are removed prior to evaporation or via
crystallization during the evaporation. For the case where salts
are removed prior to evaporation, low cost configurations are
useful. Exemplary configurations include a falling film triple
effect evaporator system or mechanical vapor recompression system.
The case where salts are crystallized during the evaporation is
more complex due to the possibility of scaling of the heat
exchanger surfaces by precipitation of the salts. An exemplary
configuration for this case includes triple effect where the first
two effects are falling film evaporators (before the onset of
crystallization) and the final stage is a forced circulation
evaporative crystallizer, for example.
[0131] 1,4-BDO purification, in particular, can occur in a series
of two distillation columns, although more can be used. A first
column is used to separate water and other light components from
1,4-BDO, while a second column is used to distill the 1,4-BDO from
any residual heavy components. The distillation columns can be
operated under vacuum to reduce the required temperatures and
reduce unwanted reactions, product degradation, and color
formation. Pressure drop across the columns can be minimized to
maintain low temperatures in the bottom reboiler. Residence time in
the reboiler can be minimized to also prevent unwanted reactions,
product degradation, and color formation, by using, for example, a
falling film reboiler.
[0132] Those skilled in the art will recognize that various
configurations of the enumerated centrifugation, filtration, ion
exchange, evaporator crystallizer, evaporator, and distillation
apparatus are useful in the purification of a compound of interest,
including 1,4-BDO. One exemplary configuration includes, for
example, disc stack centrifugation, ultrafiltration, evaporative
crystallization, ion exchange, and distillation as shown in the
flow scheme diagram of FIG. 16. Thus, in some embodiments, the
present invention provides a process of isolating 1,4-BDO from a
fermentation broth that includes removing a portion of solids by
disc stack centrifugation to provide a liquid fraction, removing a
further portion of solids from the liquid fraction by
ultrafiltration, removing a portion of salts from the liquid
fraction by evaporative crystallization, removing a further portion
of salts from the liquid fraction by ion exchange, and distilling
1,4-BDO.
[0133] As shown in FIG. 16, cells and solids are first removed by
disc stack centrifugation. The cells can be optionally recycled
back into fermentation. Ultrafiltration removes cell debris, DNA,
and precipitated proteins. Evaporative crystallization removes a
portion of the media salts and water, either of which can be
optionally recycled back into fermentation. Following evaporative
crystallization, the remaining liquid phase is passed through an
ion exchange column to remove further salts. After ion exchange, a
portion of the water can be evaporated in an evaporator system, as
described above. Distillation of the light fraction, is followed by
distillation of 1,4-BDO to provide substantially pure 1,4-BDO.
[0134] Another exemplary configuration includes disc stack
centrifugation, ultrafiltration, nanofiltration, ion exchange,
evaporation, and distillation as shown in FIG. 17. Thus, in some
embodiments, the present invention provides a process of isolating
1,4-BDO from a fermentation broth that includes removing a portion
of solids by disc stack centrifugation to provide a liquid
fraction, removing a further portion of solids from the liquid
fraction by ultrafiltration, removing a portion of salts from the
liquid fraction by nanofiltration, removing a further portion of
salts from the liquid fraction by ion exchange, evaporating a
portion of water, and distilling 1,4-BDO.
[0135] As shown in FIG. 17, cells and solids are first removed by
disc stack centrifugation. The cells can be optionally recycled
back into fermentation. Ultrafiltration removes cell debris, DNA,
and precipitated proteins. Nanofiltration removes a portion of the
media salts, which can be optionally recycled back into
fermentation. Following nanofiltration, the permeate is passed
through an ion exchange column to remove further salts. After ion
exchange, a portion of the water can be evaporated in an evaporator
system, as described above. Distillation of the light fraction, is
followed by distillation of 1,4-BDO to provide substantially pure
1,4-BDO.
[0136] The compound of interest can be any compound for which the
product can be engineered for biosynthesis in a microorganism. The
processes disclosed herein are applicable to compounds of interest
that have boiling points higher than water. Specifically, compounds
of interest can have a boiling point between about 120.degree. C.
and 400.degree. C. Other properties include high solubility or
miscibility in water and the inability to appreciably solubilize
salts (when employing evaporative crystallization), and neutral
compounds with molecular weights below about 100-150 Daltons (for
suitability with nanofiltration).
[0137] The processes and principles described herein can be applied
to isolate a compound of interest from a fermentation broth, where
the compound of interest has the general properties described
above. Such a process includes separating a liquid fraction
enriched in the compound of interest from a solid fraction that
includes the cell mass, followed by water and salt removal,
followed by purification.
[0138] In some embodiments, the invention also provides a process
for recycling components of a fermentation broth. The fermentation
broth can include 1,4-BDO or any compound of interest having a
boiling point higher than water, cells capable of producing 1,4-BDO
or the compound of interest, media salts, and water. The process
includes separating a liquid fraction enriched in 1,4-BDO or the
compound of interest from a solid fraction that includes the cells.
The cells are then recycled into the fermentation broth. Water can
be removed before or after separation of salts from the liquid
fraction. Evaporated water from the liquid fraction is recycled
into the fermentation broth. Salts from the liquid fraction can be
removed and recycled into the fermentation broth either by removal
of water from the liquid fraction, causing the salts to
crystallize, or by nanofiltration and/or ion exchange. The
separated salts from nanofiltration are then recycled into the
fermentation broth. The process provides 1,4-BDO or other compounds
of interest which can be further purified by, for example, by
distillation.
[0139] In some embodiments, a process for producing a compound of
interest, such as 1,4-BDO, includes culturing a compound-producing
microorganism in a fermentor for a sufficient period of time to
produce the compound of interest. The organism includes a
microorganism having a compound pathway comprising one or more
exogenous genes encoding a compound pathway enzyme and/or one or
more gene disruptions. The process for producing the compound also
includes isolating the compound by a process that includes
separating a liquid fraction enriched in compound of interest from
a solid fraction comprising cells, removing water from the liquid
fraction, removing salts from the liquid fraction, and purifying
the compound of interest. The compound of interest has a boiling
point higher than water.
[0140] In a specific embodiment, a process for producing 1,4-BDO
includes culturing a 1,4-BDO-producing microorganism in a fermentor
for a sufficient period of time to produce 1,4-BDO. The organism
includes a microorganism having a 1,4-BDO pathway including one or
more exogenous genes encoding a compound pathway enzyme and/or one
or more gene disruptions. The process for producing 1,4-BDO also
includes isolating the compound by a process that includes
separating a liquid fraction enriched in compound of interest from
a solid fraction comprising cells, removing water from the liquid
fraction, removing salts from the liquid fraction, and purifying
the compound of interest.
[0141] In particular embodiments where the product of interest is
1,4-BDO, production begins with the culturing of a microbial
organism capable of producing 1,4-BDO via a set of 1,4-BDO pathway
enzymes. Exemplary microbial organisms include, without limitation,
those described in U.S. 2009/0075351 and U.S. 2009/0047719, both of
which are incorporated herein by reference in their entirety.
[0142] Organisms can be provided that incorporate one or more
exogenous nucleic acids that encode enzymes in a 1,4-BDO pathway.
Such organisms include, for example, non-naturally occurring
microbial organisms engineered to have a complete 1,4-BDO
biosynthetic pathway. Such pathways can include enzymes encoded by
both endogenous and exogenous nucleic acids. Enzymes not normally
present in a microbial host can add in functionality to complete a
pathways by including one or more exogenous nucleic acids, for
example. One such 1,4-BDO pathway includes enyzmes encoding a
4-hydroxybutanoate dehydrogenase, a succinyl-CoA synthetase, a
CoA-dependent succinic semialdehyde dehydrogenase, a
4-hydroxybutyrate:CoA transferase, a 4-butyrate kinase, a
phosphotransbutyrylase, an .alpha.-ketoglutarate decarboxylase, an
aldehyde dehydrogenase, an alcohol dehydrogenase or an
aldehyde/alcohol dehydrogenase.
[0143] Another pathway can include one or more exogenous nucleic
acids encoding a 4-aminobutyrate CoA transferase, a
4-aminobutyryl-CoA hydrolase, a 4-aminobutyrate-CoA ligase, a
4-aminobutyryl-CoA oxidoreductase (deaminating), a
4-aminobutyryl-CoA transaminase, or a 4-hydroxybutyryl-CoA
dehydrogenase. Such a pathway can further include a
4-hydroxybutyryl-CoA reductase (alcohol forming), a
4-hydroxybutyryl-CoA reductase, or 1,4-butanediol dehydrogenase
[0144] Still another pathway can include one or more exogenous
nucleic acids encoding a 4-aminobutyrate CoA transferase, a
4-aminobutyryl-CoA hydrolase, a 4-aminobutyrate-CoA ligase, a
4-aminobutyryl-CoA reductase (alcohol forming), a
4-aminobutyryl-CoA reductase, a 4-aminobutan-1-ol dehydrogenase, a
4-aminobutan-1-ol oxidoreductase (deaminating) or a
4-aminobutan-1-ol transaminase. Such a pathway can further include
a 1,4-butanediol dehydrogenase.
[0145] A further pathway can include one ore more exogenous nucleic
acids encoding a 4-aminobutyrate kinase, a 4-aminobutyraldehyde
dehydrogenase (phosphorylating), a 4-aminobutan-1-ol dehydrogenase,
a 4-aminobutan-1-ol oxidoreductase (deaminating), a
4-aminobutan-1-ol transaminase, a [(4-aminobutanolyl)oxy]phosphonic
acid oxidoreductase (deaminating), a
[(4-aminobutanolyl)oxy]phosphonic acid transaminase, a
4-hydroxybutyryl-phosphate dehydrogenase, or a
4-hydroxybutyraldehyde dehydrogenase (phosphorylating). Such a
pathway can further include a 1,4-butanediol dehydrogenase.
[0146] Yet a further pathway can include one or more exogenous
nucleic acids encoding an alpha-ketoglutarate 5-kinase, a
2,5-dioxopentanoic semialdehyde dehydrogenase (phosphorylating), a
2,5-dioxopentanoic acid reductase, an alpha-ketoglutarate CoA
transferase, an alpha-ketoglutaryl-CoA hydrolase, an
alpha-ketoglutaryl-CoA ligase, an alpha-ketoglutaryl-CoA reductase,
a 5-hydroxy-2-oxopentanoic acid dehydrogenase, an
alpha-ketoglutaryl-CoA reductase (alcohol forming), a
5-hydroxy-2-oxopentanoic acid decarboxylase, or a
5-hydroxy-2-oxopentanoic acid dehydrogenase (decarboxylation). Such
a pathway can further include a 4-hydroxybutyryl-CoA reductase
(alcohol forming), a 4-hydroxybutyryl-CoA reductase, or a
1,4-butanediol dehydrogenase.
[0147] Yet a further pathway can include one or more exogenous
nucleic acids encoding a glutamate CoA transferase, a glutamyl-CoA
hydrolase, a glutamyl-CoA ligase, a glutamate 5-kinase, a
glutamate-5-semialdehyde dehydrogenase (phosphorylating), a
glutamyl-CoA reductase, a glutamate-5-semialdehyde reductase, a
glutamyl-CoA reductase (alcohol forming), a
2-amino-5-hydroxypentanoic acid oxidoreductase (deaminating), a
2-amino-5-hydroxypentanoic acid transaminase, a
5-hydroxy-2-oxopentanoic acid decarboxylase, or a
5-hydroxy-2-oxopentanoic acid dehydrogenase (decarboxylation). Such
a pathway can further include a 4-hydroxybutyryl-CoA reductase
(alcohol forming), a 4-hydroxybutyryl-CoA reductase, or a
1,4-butanediol dehydrogenase.
[0148] Yet a further pathway can include one or more exogenous
nucleic acids encoding a 3-hydroxybutyryl-CoA dehydrogenase, a
3-hydroxybutyryl-CoA dehydratase, a vinylacetyl-CoA
.DELTA.-isomerase, or a 4-hydroxybutyryl-CoA dehydratase. Such a
pathway can further include a 4-hydroxybutyryl-CoA reductase
(alcohol forming), a 4-hydroxybutyryl-CoA reductase, or a
1,4-butanediol dehydrogenase.
[0149] Yet a further pathway can include one ore more exogenous
nucleic acids encoding a homoserine deaminase, a homoserine CoA
transferase, a homoserine-CoA hydrolase, a homoserine-CoA ligase, a
homoserine-CoA deaminase, a 4-hydroxybut-2-enoyl-CoA transferase, a
4-hydroxybut-2-enoyl-CoA hydrolase, a 4-hydroxybut-2-enoyl-CoA
ligase, a 4-hydroxybut-2-enoate reductase, a 4-hydroxybutyryl-CoA
transferase, a 4-hydroxybutyryl-CoA hydrolase, a
4-hydroxybutyryl-CoA ligase, or a 4-hydroxybut-2-enoyl-CoA
reductase. Such a pathway can further include a
4-hydroxybutyryl-CoA reductase (alcohol forming), a
4-hydroxybutyryl-CoA reductase, or a 1,4-butanediol
dehydrogenase.
[0150] Yet a further pathway can include one or more exogenous
nucleic acids encoding a succinyl-CoA reductase (alcohol forming),
a 4-hydroxybutyryl-CoA hydrolase, a 4-hydroxybutyryl-CoA ligase, or
a 4-hydroxybutanal dehydrogenase (phosphorylating). Such a pathway
can further include a succinyl-CoA reductase, a 4-hydroxybutyrate
dehydrogenase, a 4-hydroxybutyryl-CoA transferase, a
4-hydroxybutyrate kinase, a phosphotrans-4-hydroxybutyrylase, a
4-hydroxybutyryl-CoA reductase, a 4-hydroxybutyryl-CoA reductase
(alcohol forming), or a 1,4-butanediol dehydrogenase.
[0151] Yet a further pathway can include one or more exogenous
nucleic acid encoding a glutamate dehydrogenase, 4-aminobutyrate
oxidoreductase (deaminating), 4-aminobutyrate transaminase,
glutamate decarboxylase, 4-hydroxybutyryl-CoA hydrolase,
4-hydroxybutyryl-CoA ligase, or 4-hydroxybutanal dehydrogenase
(phosphorylating). Such a pathway can further include an
alpha-ketoglutarate decarboxylase, a 4-hydroxybutyrate
dehydrogenase, a 4-hydroxybutyryl-CoA transferase, a
4-hydroxybutyrate kinase, a phosphotrans-4-hydroxybutyrylase, a
4-hydroxybutyryl-CoA reductase, a 4-hydroxybutyryl-CoA reductase
(alcohol forming), or a 1,4-butanediol dehydrogenase.
[0152] In addition to, or in lieu of, gene insertions, an organism
can include gene disruptions to direct the carbon flux toward the
direction of synthesizing 1,4-BDO. Such organisms include for
example, non-naturally occurring microorganism having a set of
metabolic modifications that couple 1,4-butanediol production to
growth. In some embodiments, 1,4-butanediol production is not
coupled to growth. The set of metabolic modifications can include
disruption of one or more genes, or an ortholog thereof. Disruption
can include complete gene deletion in some embodiments. Disruption
can also include modification via removal of a promoter sequence
and the like. For 1,4-BDO production a set of metabolic
modifications can include disruption of adhE and ldhA, for example.
Other disruption can include the gene mdh. Still other disruptions
can include one or more genes selected from the set of genes
including mqo, aspA, sfcA, maeB, pntAB, and gdhA. Still other
disruptions can include one or more genes selected from the set of
genes including pykA, pykF, dhaKLM, deoC, edd, yiaE, ycdW, prpC,
and gsk. Still other disruptions can include disruption of pflAB.
An exemplary set of disruptions can include one or more genes
selected from the set of genes comprising adhE, ldhA, pflAB, mdh,
and aspA, including disruption of each of the genes adhE, ldhA,
pflAB, mdh, and aspA.
[0153] Further exemplary sets of disruptions include adher, nadh6;
adher, ppck; adher, sucd4; adher, atps4r; adher, fum; adher, mdh;
adher, pfli, ppck; adher, pfli, sucd4; adher, ackr, nadh6; adher,
nadh6, pfli; adher, aspt, mdh; adher, nadh6, ppck; adher, ppck,
thd2; adher, atps4r, ppck; adher, mdh, thd2; adher, fum, pfli;
adher, ppck, sucd4; adher, glcpts, ppck; adher, gludy, mdh; adher,
gludy, ppck; adher, fum, ppck; adher, mdh, ppck; adher, fum, gludy;
adher, fum, hex1; adher, hex1, pfli; adher, hex1, thd2; adher,frd2,
ldh_d, mdh; adher,frd2, ldh_d, me2; adher, mdh, pgl, thd2; adher,
g6pdhy, mdh, thd2; adher, pfli, ppck, thd2; adher, ackr, akgd,
atps4r; adher, glcpts, pfli, ppck; adher, ackr, atps4r, sucoas;
adher, gludy, pfli, ppck; adher, me2, pfli, sucd4; adher, gludy,
pfli, sucd4; adher, atps4r, ldh_d, sucd4; adher, fum, hex1, pfli;
adher, mdh, nadh6, thd2; adher, atps4r, mdh, nadh6; adher, atps4r,
fum, nadh6; adher, aspt, mdh, nadh6; adher, aspt, mdh, thd2; adher,
atps4r, glcpts, sucd4; adher, atps4r, gludy, mdh; adher, atps4r,
mdh, ppck; adher, atps4r, fum, ppck; adher, aspt, glcpts, mdh;
adher, aspt, gludy, mdh; adher, me2, sucd4, thd2; adher, fum, ppck,
thd2; adher, mdh, ppck, thd2; adher, gludy, mdh, thd2; adher, hex1,
pfli, thd2; adher, atps4r, g6pdhy, mdh; adher, atps4r, mdh, pgl;
adher, ackr, frd2, ldh_d; adher, ackr, ldh_d, sucd4; adher, atps4r,
fum, gludy; adher, atps4r, fum, hex1; adher, atps4r, mdh, thd2;
adher, atps4r, frd2, ldh_d; adher, atps4r, mdh, pgdh; adher,
glcpts, ppck, thd2; adher, gludy, ppck, thd2; adher, fum, hex1,
thd2; adher, atps4r, me2, thd2; adher, fum, me2, thd2; adher,
glcpts, gludy, ppck; adher, me2, pgl, thd2; adher, g6pdhy, me2,
thd2; adher, atps4r, frd2, ldh_d, me2; adher, atps4r, frd2, ldh_d,
mdh; adher, aspt, ldh_d, mdh, pfli; adher, atps4r, glcpts, nadh6,
pfli; adher, atps4r, mdh, nadh6, pgl; adher, atps4r, g6pdhy, mdh,
nadh6; adher, ackr, fum, gludy, ldh_d; adher, ackr, gludy, ldh_d,
sucd4; adher, atps4r, g6pdhy, mdh, thd2; adher, atps4r, mdh, pgl,
thd2; adher, aspt, g6pdhy, mdh, pyk; adher, aspt, mdh, pgl, pyk;
adher, aspt, ldh_d, mdh, sucoas; adher, aspt, fum, ldh_d, mdh;
adher, aspt, ldh_d, mals, mdh; adher, aspt, icl, ldh_d, mdh; adher,
frd2, gludy, ldh_d, ppck; adher, frd2, ldh_d, ppck, thd2; adher,
ackr, atps4r, ldh_d, sucd4; adher, ackr, acs, ppc, ppck; adher,
gludy, ldh_d, ppc, ppck; adher, ldh_d, ppc, ppck, thd2; adher,
aspt, atps4r, glcpts, mdh; adher, g6pdhy, mdh, nadh6, thd2; adher,
mdh, nadh6, pgl, thd2; adher, atps4r, g6pdhy, glcpts, mdh; adher,
atps4r, glcpts, mdh, pgl; and adher, ackr, ldh_d, mdh, sucd4. The
aforementioned genes are included in a broader list of knockout
candidates, along with the reactions that these genes catalyze, in
Table 1a below.
TABLE-US-00001 TABLE 1a Gene Knockout Candidates in E. coli. Genes
Encoding the Enzyme(s) Reaction Catalyzing Each Abbreviation
Reaction Stoichiometry* Reaction& ACKr [c]: ac + atp <==>
actp + adp (b3115 or b2296 or b1849) ACS [c]: ac + atp + coa -->
accoa + amp + ppi b4069 ACt6 ac[p] + h[p] <==> ac[c] + h[c]
Non-gene associated ADHEr [c]: etoh + nad <==> acald + h +
nadh (b0356 or b1478 or [c]: acald + coa + nad <==> accoa + h
+ nadh b1241) (b1241 or b0351) AKGD [c]: akg + coa + nad --> co2
+ nadh + succoa (b0116 and b0726 and b0727) ASNS2 [c]: asp-L + atp
+ nh4 --> amp + asn-L + h + ppi b3744 ASPT [c]: asp-L --> fum
+ nh4 b4139 ATPS4r adp[c] + (4) h[p] + pi[c] <==> atp[c] +
(3) h[c] + h2o[c] (((b3736 and b3737 and b3738) and (b3731 and
b3732 and b3733 and b3734 and b3735)) or ((b3736 and b3737 and
b3738) and (b3731 and b3732 and b3733 and b3734 and b3735) and
b3739)) CBMK2 [c]: atp + co2 + nh4 <==> adp + cbp + (2) h
(b0521 or b0323 or b2874) EDA [c]: 2ddg6p --> g3p + pyr b1850
ENO [c]: 2pg <==> h2o + pep b2779 FBA [c]: fdp <==>
dhap + g3p (b2097 or b2925 or b1773) FBP [c]: fdp + h2o --> f6p
+ pi (b4232 or b3925) FDH2 for[p] + (2) h[c] + q8[c] --> co2[c]
+ h[p] + q8h2[c] ((b3892 and b3893 for[p] + (2) h[c] + mqn8[c]
--> co2[c] + h[p] + mql8[c] and b3894) or (b1474 and b1475 and
b1476)) FRD2 [c]: fum + mql8 --> mqn8 + succ (b4151 and b4152
[c]: 2dmmql8 + fum --> 2dmmq8 + succ and b4153 and b4154) FTHFD
[c]: 10fthf + h2o --> for + h + thf b1232 FUM [c]: fum + h2o
<==> mal-L (b1612 or b4122 or b1611) G5SD [c]: glu5p + h +
nadph --> glu5sa + nadp + pi b0243 G6PDHy [c]: g6p + nadp
<==> 6pgl + h + nadph b1852 GLCpts glc-D[p] + pep[c] -->
g6p[c] + pyr[c] ((b2417 and b1101 and b2415 and b2416) or (b1817
and b1818 and b1819 and b2415 and b2416) or (b2417 and b1621 and
b2415 and b2416)) GLU5K [c]: atp + glu-L --> adp + glu5p b0242
GLUDy [c]: glu-L + h2o + nadp <==> akg + h + nadph + nh4
b1761 GLYCL [c]: gly + nad + thf --> co2 + mlthf + nadh + nh4
(b2904 and b2903 and b2905 and b0116) HEX1 [c]: atp + glc-D -->
adp + g6p + h b2388 ICL [c]: icit --> glx + succ b4015 LDH_D
[c]: lac-D + nad <==> h + nadh + pyr (b2133 or b1380) MALS
[c]: accoa + glx + h2o --> coa + h + mal-L (b4014 or b2976) MDH
[c]: mal-L + nad <==> h + nadh + oaa b3236 ME2 [c]: mal-L +
nadp --> co2 + nadph + pyr b2463 MTHFC [c]: h2o + methf
<==> 10fthf + h b0529 NADH12 [c]: h + mqn8 + nadh --> mql8
+ nad b1109 [c]: h + nadh + q8 --> nad + q8h2 [c]: 2dmmq8 + h +
nadh --> 2dmmql8 + nad NADH6 (4) h[c] + nadh[c] + q8[c] -->
(3) h[p] + nad[c] + q8h2[c] (b2276 and b2277 (4) h[c] + mqn8[c] +
nadh[c] --> (3) h[p] + mql8[c] + and b2278 and b2279 nad[c] and
b2280 and b2281 2dmmq8[c] + (4) h[c] + nadh[c] --> 2dmmql8[c] +
(3) and b2282 and b2283 h[p] + nad[c] and b2284 and b2285 and b2286
and b2287 and b2288) PFK [c]: atp + f6p --> adp + fdp + h (b3916
or b1723) PFLi [c]: coa + pyr --> accoa + for (((b0902 and
b0903) and b2579) or (b0902 and b0903) or (b0902 and b3114) or
(b3951 and b3952)) PGDH [c]: 6pgc + nadp --> co2 + nadph +
ru5p-D b2029 PGI [c]: g6p <==> f6p b4025 PGL [c]: 6pgl + h2o
--> 6pgc + h b0767 PGM [c]: 2pg <==> 3pg (b3612 or b4395
or b0755) PPC [c]: co2 + h2o + pep --> h + oaa + pi b3956 PPCK
[c]: atp + oaa --> adp + co2 + pep b3403 PRO1z [c]: fad + pro-L
--> 1pyr5c + fadh2 + h b1014 PYK [c]: adp + h + pep --> atp +
pyr b1854 or b1676) PYRt2 h[p] + pyr[p] <==> h[c] + pyr[c]
Non-gene associated RPE [c]: ru5p-D <==> xu5p-D (b4301 or
b3386) SO4t2 so4[e] <==> so4[p] (b0241 or b0929 or b1377 or
b2215) SUCD4 [c]: q8 + succ --> fum + q8h2 (b0721 and b0722 and
b0723 and b0724) SUCOAS [c]: atp + coa + succ <==> adp + pi +
succoa (b0728 and b0729) SULabc atp[c] + h2o[c] + so4[p] -->
adp[c] + h[c] + pi[c] + ((b2422 and b2425 so4[c] and b2424 and
b2423) or (b0763 and b0764 and b0765) or (b2422 and b2424 and b2423
and b3917)) TAL [c]: g3p + s7p <==> e4p + f6p (b2464 or
b0008) THD2 (2) h[p] + nadh[c] + nadp[c] --> (2) h[c] + nad[c] +
(b1602 and b1603) nadph[c] THD5 [c]: nad + nadph --> nadh + nadp
(b3962 or (b1602 and b1603)) TPI [c]: dhap <==> g3p b3919
[0154] The abbreviations for the metabolites in Table 1a are shown
below in Table 1b.
TABLE-US-00002 TABLE 1b Metabolite names corresponding to
abbreviations used in Table 1a. Metabolite Abbreviation Metabolite
Name 10fthf 10-Formyltetrahydrofolate 1pyr5c
1-Pyrroline-5-carboxylate 2ddg6p 2-Dehydro-3-deoxy-D-gluconate
6-phosphate 2dmmq8 2-Demethylmenaquinone 8 2dmmql8
2-Demethylmenaquinol 8 2pg D-Glycerate 2-phosphate 3pg
3-Phospho-D-glycerate 6pgc 6-Phospho-D-gluconate 6pgl
6-phospho-D-glucono-1,5-lactone ac Acetate acald Acetaldehyde accoa
Acetyl-CoA actp Acetyl phosphate adp ADP akg 2-Oxoglutarate amp AMP
asn-L L-Asparagine asp-L L-Aspartate atp ATP cbp Carbamoyl
phosphate co2 CO2 coa Coenzyme A dhap Dihydroxyacetone phosphate
e4p D-Erythrose 4-phosphate etoh Ethanol f6p D-Fructose 6-phosphate
fad Flavin adenine dinucleotide oxidized fadh2 Flavin adenine
dinucleotide reduced fdp D-Fructose 1,6-bisphosphate for Formate
fum Fumarate g3p Glyceraldehyde 3-phosphate g6p D-Glucose
6-phosphate glc-D D-Glucose glu5p L-Glutamate 5-phosphate glu5sa
L-Glutamate 5-semialdehyde glu-L L-Glutamate glx Glyoxylate gly
Glycine h H+ h2o H2O icit Isocitrate lac-D D-Lactate mal-L L-Malate
methf 5,10-Methenyltetrahydrofolate mlthf
5,10-Methylenetetrahydrofolate mql8 Menaquinol 8 mqn8 Menaquinone 8
nad Nicotinamide adenine dinucleotide nadh Nicotinamide adenine
dinucleotide - reduced nadp Nicotinamide adenine dinucleotide
phosphate nadph Nicotinamide adenine dinucleotide phosphate -
reduced nh4 Ammonium oaa Oxaloacetate pep Phosphoenolpyruvate pi
Phosphate ppi Diphosphate pro-L L-Proline pyr Pyruvate q8
Ubiquinone-8 q8h2 Ubiquinol-8 ru5p-D D-Ribulose 5-phosphate s7p
Sedoheptulose 7-phosphate so4 Sulfate succ Succinate succoa
Succinyl-CoA thf 5,6,7,8-Tetrahydrofolate xu5p-D D-Xylulose
5-phosphate
[0155] Any non-naturally occurring microorganism incorporating any
combination of the above gene disruptions can also include a gene
insertion of at least one exogenous nucleic acid. Any of the gene
insertion pathways described above can be integrated with gene
disruptions. For example, a pathway including disruptions of the
genes adhE, ldhA, pflAB, mdh, and aspA can also include insertion
of a 4-hydroxybutanoate dehydrogenase, a CoA-independent succinic
semialdehyde dehydrogenase, a succinyl-CoA synthetase, a
CoA-dependent succinic semialdehyde dehydrogenase, a
4-hydroxybutyrate:CoA transferase, a glutamate:succinic
semialdehyde transaminase, a glutamate decarboxylase, a
CoA-independent aldehyde dehydrogenase, a CoA-dependent aldehyde
dehydrogenase or an alcohol dehydrogenase. Table 2 below summarizes
exemplary engineered organisms for the production of 1,4-BDO that
incorporate combinations of gene disruption and gene insertion.
Note that gene insertion can be in the form of chromosomal
insertion or providing a plasmid.
TABLE-US-00003 TABLE 2 Combination Disruption-Insertion Designs for
1,4-BDO Production Strain # Host chromosome Host Description
Plasmid-based 1 .DELTA.ldhA Single deletion E. coli sucCD, P.
gingivalis sucD, derivative of E. coli P. gingivalis 4hbd, P.
gingivalis MG1655 Cat2, C. acetobutylicum AdhE2 2 .DELTA.adhE
.DELTA.ldhA .DELTA.pflB Succinate producing E. coli sucCD, P.
gingivalis sucD, strain; derivative of P. gingivalis 4hbd, P.
gingivalis E. coli MG1655 Cat2, C. acetobutylicum AdhE2 3
.DELTA.adhE .DELTA.ldhA .DELTA.pflB Improvement of lpdA to E. coli
sucCD, P. gingivalis sucD, .DELTA.lpdA::K.p.lpdA322 increase
pyruvate P. gingivalis 4hbd, P. gingivalis dehydrogenase flux Cat2,
C. acetobutylicum AdhE2 4 .DELTA.adhE .DELTA.ldhA .DELTA.pflB E.
coli sucCD, P. gingivalis sucD, .DELTA.lpdA::K.p.lpdA322 P.
gingivalis 4hbd, C. acetobutylicum buk1, C. acetobutylicum ptb, C.
acetobutylicum AdhE2 5 .DELTA.adhE .DELTA.ldhA .DELTA.pflB
Deletions in mdh and E. coli sucCD, P. gingivalis sucD,
.DELTA.lpdA::K.p.lpdA322 .DELTA.mdh .DELTA.arcA arcA to direct flux
P. gingivalis 4hbd, P. gingivalis through oxidative TCA Cat2, C.
acetobutylicum AdhE2 cycle 6 .DELTA.adhE .DELTA.ldhA .DELTA.pflB M.
bovis sucA, E. coli sucCD, .DELTA.lpdA::K.p.lpdA322 .DELTA.mdh
.DELTA.arcA P. gingivalis sucD, P. gingivalis 4hbd, P. gingivalis
Cat2, C. acetobutylicum AdhE2 7 .DELTA.adhE .DELTA.ldhA .DELTA.pflB
Mutation in citrate E. coli sucCD, P. gingivalis sucD,
.DELTA.lpdA::K.p.lpdA322 .DELTA.mdh .DELTA.arcA synthase to improve
P. gingivalis 4hbd, P. gingivalis gltAR163L anaerobic activity
Cat2, C. acetobutylicum AdhE2 8 .DELTA.adhE .DELTA.ldhA .DELTA.pflB
M. bovis sucA, E. coli sucCD, .DELTA.lpdA::K.p.lpdA322 .DELTA.mdh
.DELTA.arcA P. gingivalis sucD, P. gingivalis 4hbd, gltAR163L P.
gingivalis Cat2, C. acetobutylicum AdhE2 9 .DELTA.adhE .DELTA.ldhA
.DELTA.pflB M. bovis sucA, E. coli sucCD, .DELTA.lpdA::K.p.lpdA322
.DELTA.mdh .DELTA.arcA P. gingivalis sucD, P. gingivalis 4hbd,
gltAR163L P. gingivalis Cat2, C. beijerinckii Ald 10 .DELTA.adhE
.DELTA.ldhA .DELTA.pflB Succinate branch of P. gingivalis Cat2,
.DELTA.lpdA::K.p.lpdA322 .DELTA.mdh .DELTA.arcA upstream pathway C.
beijerinckii gltAR163L fimD:: E. coli sucCD, integrated into ECKh-
Ald P. gingivalis sucD, P. gingivalis 4hbd 422 11 .DELTA.adhE
.DELTA.ldhA .DELTA.pflB .DELTA.lpdA::K.p.lpdA322 .DELTA.mdh
Succinate and alpha- P. gingivalis Cat2, .DELTA.arcA gltAR163L
fimD:: E. coli sucCD, ketoglutarate upstream C. beijerinckii Ald P.
gingivalis sucD, P. gingivalis 4hbd fimD:: pathway branches M.
bovis sucA, C. kluyveri 4hbd integrated into ECKh-422 12
.DELTA.adhE .DELTA.ldhA .DELTA.pflB .DELTA.lpdA::K.p.lpdA322
.DELTA.mdh C. acetobutylicum buk1, .DELTA.arcA gltAR163L fimD:: E.
coli sucCD, C. acetobutylicum ptb, P. gingivalis sucD, P.
gingivalis 4hbd fimD:: C. beijerinckii Ald M. bovis sucA, C.
kluyveri 4hbd 13 .DELTA.adhE .DELTA.ldhA .DELTA.pflB
.DELTA.lpdA::K.p.lpdA322 .DELTA.mdh Acetate kinase deletion of P.
gingivalis Cat2, .DELTA.arcA gltAR163L .DELTA.ackA fimD:: E. coli
sucCD, ECKh-432 C. beijerinckii Ald P. gingivalis sucD, P.
gingivalis 4hbd fimD:: M. bovis sucA, C. kluyveri 4hbd 14
.DELTA.adhE .DELTA.ldhA .DELTA.pflB .DELTA.lpdA::K.p.lpdA322
.DELTA.mdh Acetate kinase deletion P. gingivalis Cat2, .DELTA.arcA
gltAR163L .DELTA.ackA .DELTA.ppc::H.i.ppck fimD:: and PPC/PEPCK C.
beijerinckii Ald E. coli sucCD, P. gingivalis sucD, P. gingivalis
replacement of ECKh- 4hbd fimD:: M. bovis sucA, C. kluyveri 4hbd
432 15 .DELTA.adhE .DELTA.ldhA .DELTA.pflB .DELTA.lpdA::fnr-pflB6-
Replacement of lpdA P. gingivalis Cat2, K.p.lpdA322 .DELTA.mdh
.DELTA.arcA gltAR163L fimD:: promoter with anaerobic C.
beijerinckii Ald E. coli sucCD, P. gingivalis sucD, P. gingivalis
4hbd promoter in ECKh-432 fimD:: M. bovis sucA, C. kluyveri 4hbd 16
.DELTA.adhE .DELTA.ldhA .DELTA.pflB .DELTA.lpdA:: K.p.lpdA322
Replacement of pdhR and P. gingivalis Cat2, .DELTA.pdhR:: fnr-pflB6
.DELTA.mdh .DELTA.arcA gltAR163L aceEF promoter with C.
beijerinckii Ald fimD:: E. coli sucCD, P. gingivalis sucD,
anaerobic promoter in P. gingivalis 4hbd fimD:: M. bovis sucA, C.
kluyveri ECKh-432 4hbd 17 .DELTA.adhE .DELTA.ldhA .DELTA.pflB
.DELTA.lpdA:: K.p.lpdA322 .DELTA.mdh Integration of BK/PTB C.
beijerinckii Ald .DELTA.arcA gltAR163L fimD:: E. coli sucCD, into
ECKh-432 P. gingivalis sucD, P. gingivalis 4hbd fimD:: M. bovis
sucA, C. kluyveri 4hbd fimD:: C. acetobutylicum buk1, C.
acetobutylicum ptb 18 .DELTA.adhE .DELTA.ldhA .DELTA.pflB
.DELTA.lpdA:: K.p.lpdA322 .DELTA.mdh C. beijerinckii Ald,
.DELTA.arcA gltAR163L fimD:: E. coli sucCD, G. thermoglucosidasius
adh1 P. gingivalis sucD, P. gingivalis 4hbd fimD:: M. bovis sucA,
C. kluyveri 4hbd fimD:: C. acetobutylicum buk1, C. acetobutylicum
ptb 19 .DELTA.adhE .DELTA.ldhA .DELTA.pflB .DELTA.lpdA::K.p.lpdA322
.DELTA.mdh Non-PTS sucrose genes P. gingivalis Cat2, .DELTA.arcA
gltAR163L fimD:: E. coli sucCD, inserted into ECKh-432 C.
beijerinckii Ald P. gingivalis sucD, P. gingivalis 4hbd fimD:: M.
bovis sucA, C. kluyveri 4hbd rrnC::cscAKB 20 .DELTA.adhE
.DELTA.ldhA .DELTA.pflB .DELTA.lpdA::K.p.lpdA322 .DELTA.mdh C.
acetobutylicum buk1, .DELTA.arcA gltAR163L fimD:: E. coli sucCD, C.
acetobutylicum ptb, P. gingivalis sucD, P. gingivalis 4hbd fimD::
C. beijerinckii Ald M. bovis sucA, C. kluyveri 4hbd rrnC::cscAKB
*The delta symbol (.DELTA.) indicates gene deletion.
[0156] The strains summarized in Table 2 are as follows: Strain 1:
Single deletion derivative of E. coli MG1655, with deletion of
endogenous ldhA; plasmid expression of E. coli sucCD, P. gingivalis
sucD, P. gingivalis 4hbd, P. gingivalis Cat2, C. acetobutylicum
AdhE2. Strain 2: Host strain AB3, a succinate producing strain,
derivative of E. coli MG1655, with deletions of endogenous adhE
ldhA pflB; plasmid expression of E. coli sucCD, P. gingivalis sucD,
P. gingivalis 4hbd, P. gingivalis Cat2, C. acetobutylicum
AdhE2.
[0157] Strain 3: Host strain ECKh-138, deletion of endogenous adhE,
ldhA, pflB, deletion of endogenous lpdA and chromosomal insertion
of Klebsiella pneumoniae lpdA with a Glu354Lys mutation at the lpdA
locus; plasmid expression of E. coli sucCD, P. gingivalis sucD, P.
gingivalis 4hbd, P. gingivalis Cat2, C. acetobutylicum AdhE2;
strain provides improvement of 1pdA to increase pyruvate
dehydrogenase flux. Strain 4: Host strain ECKh-138, deletion of
endogenous adhE, ldhA, pflB, and lpdA, chromosomal insertion of
Klebsiella pneumoniae lpdA with a Glu354Lys mutation; plasmid
expression E. coli sucCD, P. gingivalis sucD, P. gingivalis 4hbd,
C. acetobutylicum buk1, C. acetobutylicum ptb, C. acetobutylicum
AdhE2.
[0158] Strain 5: Host strain ECKh-401, deletion of endogenous adhE,
ldhA, pflB, deletion of endogenous lpdA and chromosomal insertion
of Klebsiella pneumoniae lpdA with a Glu354Lys mutation at the lpdA
locus, deletion of endogenous mdh and arcA; plasmid expression of
E. coli sucCD, P. gingivalis sucD, P. gingivalis 4hbd, P.
gingivalis Cat2, C. acetobutylicum AdhE2; strain has deletions in
mdh and arcA to direct flux through oxidative TCA cycle. Strain 6:
host strain ECKh-401, deletion of endogenous adhE, ldhA, pflB,
deletion of endogenous lpdA and chromosomal insertion of Klebsiella
pneumoniae lpdA with a Glu354Lys mutation at the lpdA locus,
deletion of endogenous mdh and arcA; plasmid expression of M. Bovis
sucA, E. coli sucCD, P. gingivalis sucD, P. gingivalis 4hbd, P.
gingivalis Cat2, C. acetobutylicum AdhE2.
[0159] Strain 7: Host strain ECKh-422, deletion of endogenous adhE,
ldhA, pflB, deletion of endogenous lpdA and chromosomal insertion
of Klebsiella pneumoniae lpdA with a Glu354Lys mutation at the lpdA
locus, deletion of endogenous mdh and arcA, chromosomal replacement
of gltA with gltA Arg163Leu mutant; plasmid expression of E. coli
sucCD, P. gingivalis sucD, P. gingivalis 4hbd, P. gingivalis Cat2,
C. acetobutylicum AdhE2; strain has mutation in citrate synthase to
improve anaerobic activity. Strain 8: strain ECKh-422, deletion of
endogenous adhE, ldhA, pflB, deletion of endogenous lpdA and
chromosomal insertion of Klebsiella pneumoniae lpdA with a
Glu354Lys mutation at the lpdA locus, deletion of endogenous mdh
and arcA, chromosomal replacement of gltA with gltA Arg163Leu
mutant; plasmid expression of M. bovis sucA, E. coli sucCD, P.
gingivalis sucD, P. gingivalis 4hbd, P. gingivalis Cat2, C.
acetobutylicum AdhE2. Strain 9: host strain ECKh-422, deletion of
endogenous adhE, ldhA, pflB, deletion of endogenous lpdA and
chromosomal insertion of Klebsiella pneumoniae lpdA with a
Glu354Lys mutation at the lpdA locus, deletion of endogenous mdh
and arcA, chromosomal replacement of gltA with gltA Arg163Leu
mutant; plasmid expression of M. bovis sucA, E. coli sucCD, P.
gingivalis sucD, P. gingivalis 4hbd, P. gingivalis Cat2, C.
beijerinckii Ald.
[0160] Strain 10: host strain ECKh-426, deletion of endogenous
adhE, ldhA, pflB, deletion of endogenous lpdA and chromosomal
insertion of Klebsiella pneumoniae lpdA with a Glu354Lys mutation
at the lpdA locus, deletion of endogenous mdh and arcA, chromosomal
replacement of gltA with gltA Arg163Leu mutant, chromosomal
insertion at the fimD locus of E. coli sucCD, P. gingivalis sucD,
P. gingivalis 4hbd; plasmid expression of P. gingivalis Cat2, C.
beijerinckii Ald; strain has succinate branch of upstream pathway
integrated into strain ECKh-422 at the fimD locus. Strain 11: host
strain ECKh-432, deletion of endogenous adhE, ldhA, pflB, deletion
of endogenous lpdA and chromosomal insertion of Klebsiella
pneumoniae lpdA with a Glu354Lys mutation at the lpdA locus,
deletion of endogenous mdh and arcA, chromosomal replacement of
gltA with gltA Arg163Leu mutant, chromosomal insertion at the fimD
locus of E. coli sucCD, P. gingivalis sucD, P. gingivalis 4hbd,
chromosomal insertion at the fimD locus of M. bovis sucA, C.
kluyveri 4hbd; plasmid expression of P. gingivalis Cat2, C.
beijerinckii Ald; strain has succinate and alpha-ketoglutarate
upstream pathway branches integrated into ECKh-422. Strain 12: host
strain ECKh-432, deletion of endogenous adhE, ldhA, pflB, deletion
of endogenous lpdA and chromosomal insertion of Klebsiella
pneumoniae lpdA with a Glu354Lys mutation at the lpdA locus,
deletion of endogenous mdh and arcA, chromosomal replacement of
gltA with gltA Arg163Leu mutant, chromosomal insertion at the fimD
locus of E. coli sucCD, P. gingivalis sucD, P. gingivalis 4hbd,
chromosomal insertion at the fimD locus of M. bovis sucA, C.
kluyveri 4hbd; plasmid expression of C. acetobutylicum buk1, C.
acetobutylicum ptb, C. beijerinckii Ald.
[0161] Strain 13: host strain ECKh-439, deletion of endogenous
adhE, ldhA, pflB, deletion of endogenous lpdA and chromosomal
insertion of Klebsiella pneumoniae lpdA with a Glu354Lys mutation
at the lpdA locus, deletion of endogenous mdh and arcA, chromosomal
replacement of gltA with gltA Arg163Leu mutant, deletion of
endogenous ackA, chromosomal insertion at the fimD locus of E. coli
sucCD, P. gingivalis sucD, P. gingivalis 4hbd, chromosomal
insertion at the fimD locus of M. bovis sucA, C. kluyveri 4hbd;
plasmid expression of P. gingivalis Cat2, C. beijerinckii Ald;
strain has acetate kinase deletion in strain ECKh-432. Strain 14:
host strain ECKh-453, deletion of endogenous adhE, ldhA, pflB,
deletion of endogenous lpdA and chromosomal insertion of Klebsiella
pneumoniae lpdA with a Glu354Lys mutation at the lpdA locus,
deletion of endogenous mdh and arcA, chromosomal replacement of
gltA with gltA Arg163Leu mutant, deletion of endogenous ackA,
deletion of endogenous ppc and insertion of Haemophilus influenza
ppck at the ppc locus, chromosomal insertion at the fimD locus of
E. coli sucCD, P. gingivalis sucD, P. gingivalis 4hbd, chromosomal
insertion at the fimD locus of M. bovis sucA, C. kluyveri 4hbd;
plasmid expression of P. gingivalis Cat2, C. beijerinckii Ald;
strain has acetate kinase deletion and PPC/PEPCK replacement in
strain ECKh-432.
[0162] Strain 15: host strain ECKh-456, deletion of endogenous
adhE, ldhA, pflB, deletion of endogenous lpdA and chromosomal
insertion of Klebsiella pneumoniae lpdA with a Glu354Lys mutation
at the lpdA locus, deletion of endogenous mdh and arcA, chromosomal
replacement of gltA with gltA Arg163Leu mutant, chromosomal
insertion at the fimD locus of E. coli sucCD, P. gingivalis sucD,
P. gingivalis 4hbd, chromosomal insertion at the fimD locus of M.
bovis sucA, C. kluyveri 4hbd, replacement of lpdA promoter with fnr
binding site, pflB-p6 promoter and RBS of pflB; plasmid expression
of P. gingivalis Cat2, C. beijerinckii Ald; strain has replacement
of lpdA promoter with anaerobic promoter in strain ECKh-432. Strain
16: host strain ECKh-455, deletion of endogenous adhE, ldhA, pflB,
deletion of endogenous lpdA and chromosomal insertion of Klebsiella
pneumoniae lpdA with a Glu354Lys mutation at the lpdA locus,
deletion of endogenous mdh and arcA, chromosomal replacement of
gltA with gltA Arg163Leu mutant, chromosomal insertion at the fimD
locus of E. coli sucCD, P. gingivalis sucD, P. gingivalis 4hbd,
chromosomal insertion at the fimD locus of M. bovis sucA, C.
kluyveri 4hbdI, replacement of pdhR and aceEF promoter with fnr
binding site, pflB-p6 promoter and RBS of pflB; plasmid expression
of P. gingivalis Cat2, C. beijerinckii Ald; strain has replacement
of pdhR and aceEF promoter with anaerobic promoter in ECKh-432.
[0163] Strain 17: host strain ECKh-459, deletion of endogenous
adhE, ldhA, pflB, deletion of endogenous lpdA and chromosomal
insertion of Klebsiella pneumoniae lpdA with a Glu354Lys mutation
at the lpdA locus, deletion of endogenous mdh and arcA, chromosomal
replacement of gltA with gltA Arg163Leu mutant, chromosomal
insertion at the fimD locus of E. coli sucCD, P. gingivalis sucD,
P. gingivalis 4hbd, chromosomal insertion at the fimD locus of M.
bovis sucA, C. kluyveri 4hbd, chromosomal insertion at the fimD
locus of C. acetobutylicum buk1, C. acetobutylicum ptb; plasmid
expression of C. beijerinckii Ald; strain has integration of BK/PTB
into strain ECKh-432. Strain 18: host strain ECKh-459, deletion of
endogenous adhE, ldhA, pflB, deletion of endogenous lpdA and
chromosomal insertion of Klebsiella pneumoniae lpdA with a
Glu354Lys mutation at the lpdA locus, deletion of endogenous mdh
and arcA, chromosomal replacement of gltA with gltA Arg163Leu
mutant, chromosomal insertion at the fimD locus of E. coli sucCD,
P. gingivalis sucD, P. gingivalis 4hbd, chromosomal insertion at
the fimD locus of M. bovis sucA, C. kluyveri 4hbd, chromosomal
insertion at the fimD locus of C. acetobutylicum buk1, C.
acetobutylicum ptb; plasmid expression of C. beijerinckii Ald, G.
thermoglucosidasius adh1.
[0164] Strain 19: host strain ECKh-463, deletion of endogenous
adhE, ldhA, pflB, deletion of endogenous lpdA and chromosomal
insertion of Klebsiella pneumoniae lpdA with a Glu354Lys mutation
at the lpdA locus, deletion of endogenous mdh and arcA, chromosomal
replacement of gltA with gltA Arg163Leu mutant, chromosomal
insertion at the fimD locus of E. coli sucCD, P. gingivalis sucD,
P. gingivalis 4hbd, chromosomal insertion at the fimD locus of M.
bovis sucA, C. kluyveri 4hbd, insertion at the rrnC locus of
non-PTS sucrose operon genes sucrose permease (cscB),
D-fructokinase (cscK), sucrose hydrolase (cscA), and a LacI-related
sucrose-specific repressor (cscR); plasmid expression of P.
gingivalis Cat2, C. beijerinckii Ald; strain has non-PTS sucrose
genes inserted into strain ECKh-432. Strain 20: host strain
ECKh-463 deletion of endogenous adhE, ldhA, pflB, deletion of
endogenous lpdA and chromosomal insertion of Klebsiella pneumoniae
lpdA with a Glu354Lys mutation at the lpdA locus, deletion of
endogenous mdh and arcA, chromosomal replacement of gltA with gltA
Arg163Leu mutant, chromosomal insertion at the fimD locus of E.
coli sucCD, P. gingivalis sucD, P. gingivalis 4hbd, chromosomal
insertion at the fimD locus of M. bovis sucA, C. kluyveri 4hbd,
insertion at the rrnC locus of non-PTS sucrose operon; plasmid
expression of C. acetobutylicum buk1, C. acetobutylicum ptb, C.
beijerinckii Ald.
[0165] Strains engineered for the utilization of sucrose via a
phosphotransferase (PTS) system produce significant amounts of
pyruvate as a byproduct. Therefore, the use of a non-PTS sucrose
system can be used to decrease pyruvate formation because the
import of sucrose would not be accompanied by the conversion of
phosphoenolpyruvate (PEP) to pyruvate. This will increase the PEP
pool and the flux to oxaloacetate through PPC or PEPCK.
[0166] Insertion of a non-PTS sucrose operon into the rrnC region
can be performed. To generate a PCR product containing the non-PTS
sucrose genes flanked by regions of homology to the rrnC region,
two oligos are used to PCR amplify the csc genes from Mach1.TM.
(Invitrogen, Carlsbad, Calif.). This strain is a descendent of W
strain which is an E. coli strain known to be able to catabolize
sucrose (Orencio-Trejo et al., Biotechnology Biofuels 1:8 (2008)).
The sequence was derived from E. coli W strain KO11 (accession
AY314757) (Shukla et al., Biotechnol. Lett. 26:689-693 (2004)) and
includes genes encoding a sucrose permease (cscB), D-fructokinase
(cscK), sucrose hydrolase (cscA), and a LacI-related
sucrose-specific repressor (cscR). The first 53 amino acids of cscR
was effectively removed by the placement of the primer. After
purification, the PCR product is electroporated into MG1655
electrocompetent cells which had been transformed with pRedET (tet)
and prepared according to the manufacturer's instructions
(www.genebridges.com/gb/pdf/K001%20Q%20E%20BAC%20Modification%20Kit-versi-
on2.6-2007-screen.pdf). The PCR product is designed so that it
integrates into the genome into the rrnC region of the chromosome.
It effectively deletes 191 nucleotides upstream of rrlC (23S rRNA),
all of the rrlC rRNA gene and 3 nucleotides downstream of rrlC and
replaces it with the sucrose operon. The entire rrnC::crcAKB region
is transferred into the BDO host strain ECKh-432 by P1 transduction
(Sambrook et al., Molecular Cloning: A Laboratory Manual, Third
Ed., Cold Spring Harbor Laboratory, New York (2001), resulting in
ECKh-463 (.DELTA.adhE .DELTA.ldhA .DELTA.pflB
.DELTA.lpdA::K.p.lpdA322 .DELTA.mdh .DELTA.arcA gltAR163L fimD:: E.
coli sucCD, P. gingivalis sucD, P. gingivalis 4hbd fimD:: M. bovis
sucA, C. kluyveri 4hbd rrnC::cscAKB). Recombinants are selected by
growth on sucrose and verified by diagnostic PCR.
[0167] Prior to culturing the compound-producing or
1,4-BDO-producing organisms, the raw materials feedstock such as
sucrose syrup and media components can be treated, for example, by
heat sterilization prior to addition to the production bioreactor
to eliminate any biological contaminants. In accordance with some
embodiments, the feedstock can include, for example, sucrose or
glucose for the fermentation of BDO. In some embodiments, the
feedstock can include syngas. Additional media components used to
support growth of the microorganisms include, for example, salts,
nitrogen sources, buffers, trace metals, and a base for pH control.
The major components of an exemplary media package, expressed in
g/L of fermentation broth, are shown below in Table 3.
TABLE-US-00004 TABLE 3 Category Concentration N-Source 3 g/L Buffer
5 g/L Salts 0.65 g/L Base 1.4 g/L 10.1 g/L
[0168] The type of carbon source can vary considerably and can
include glucose, fructose, lactose, sucrose, maltodextrins, starch,
inulin, glycerol, vegetable oils such as soybean oil, hydrocarbons,
alcohols such as methanol and ethanol, organic acids such as
acetate, syngas, and similar combinations of CO, CO.sub.2, and
H.sub.2. The term "glucose" includes glucose syrups, i.e. glucose
compositions comprising glucose oligomers. Plant and plant-derived
biomass material can be a source of low cost feedstock. Such
feedstock can include, for example, corn, soybeans, cotton,
flaxseed, rapeseed, sugar cane and palm oil. Biomass can undergo
enzyme or chemical mediated hydrolysis to liberate substrates which
can be further processed via biocatalysis to produce chemical
products of interest. These substrates include mixtures of
carbohydrates, as well as aromatic compounds and other products
that are collectively derived from the cellulosic, hemicellulosic,
and lignin portions of the biomass. The carbohydrates generated
from the biomass are a rich mixture of 5 and 6 carbon sugars that
include, for example, sucrose, glucose, xylose, arabinose,
galactose, mannose, and fructose.
[0169] The carbon source can be added to the culture as a solid,
liquid, or gas. The carbon source can be added in a controlled
manner to avoid stress on the cells due to overfeeding. In this
respect, fed-batch and continuous culturing are useful culturing
modes as further discussed below.
[0170] The type of nitrogen source can vary considerably and can
include urea, ammonium hydroxide, ammonium salts, such as ammonium
sulphate, ammonium phosphate, ammonium chloride and ammonium
nitrate, other nitrates, amino acids such as glutamate and lysine,
yeast extract, yeast autolysates, yeast nitrogen base, protein
hydrolysates (including, but not limited to, peptones, casein
hydrolysates such as tryptone and casamino acids), soybean meal,
Hy-Soy, tryptic soy broth, cotton seed meal, malt extract, corn
steep liquor and molasses.
[0171] The pH of the culture can be controlled by the addition of
acid or alkali. Because pH can drop during culture, alkali can be
added as necessary. Examples of suitable alkalis include NaOH and
NH.sub.4OH.
[0172] Exemplary cell growth procedures used in the production of a
compound of interest, such as 1,4-BDO, include, batch fermentation,
fed-batch fermentation with batch separation; fed-batch
fermentation with continuous separation, and continuous
fermentation with continuous separation. All of these processes are
well known in the art. Depending on the organism design, the
fermentations can be carried out under aerobic or anaerobic
conditions. In some embodiments, the temperature of the cultures
kept between about 30 and about 45.degree. C., including 31, 32,
33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, and 44.degree. C.
[0173] In batch fermentation, a tank fermenter (or bioreactor) is
filled with the prepared media to support growth. The temperature
and pH for microbial fermentation is properly adjusted, and any
additional supplements are added. An inoculum of a
1,4-BDO-producing organism is added to the fermenter. In batch
fermentation the fermentation will generally run for a fixed period
and then the products from the fermentation are isolated. The
process can be repeated in batch runs.
[0174] In fed-batch fermentation fresh media is continuously or
periodically added to the fermentation bioreactor. Fixed-volume
fed-batch fermentation is a type of fed-batch fermentation in which
a carbon source is fed without diluting the culture. The culture
volume can also be maintained nearly constant by feeding the growth
carbon source as a concentrated liquid or gas. In another type of
fixed-volume fed-batch culture, sometimes called a cyclic fed-batch
culture, a portion of the culture is periodically withdrawn and
used as the starting point for a further fed-batch process. Once
the fermentation reaches a certain stage, the culture is removed
and the biomass is diluted to the original volume with sterile
water or medium containing the carbon feed substrate. The dilution
decreases the biomass concentration and results in an increase in
the specific growth rate. Subsequently, as feeding continues, the
growth rate will decline gradually as biomass increases and
approaches the maximum sustainable in the vessel once more, at
which point the culture can be diluted again. Alternatively, a
fed-batch fermentation can be variable volume. In variable-volume
mode the volume of the fermentation broth changes with the
fermentation time as nutrient and media are continually added to
the culture without removal of a portion of the fermentation
broth.
[0175] In a continuous fermentation, fresh media is generally
continually added with continuous separation of spent medium, which
can include the product of interest, such as 1,4-BDO, when the
product is secreted. One feature of the continuous culture is that
a time-independent steady-state can be obtained which enables one
to determine the relations between microbial behavior and the
environmental conditions. Achieving this steady-state is
accomplished by means of a chemostat, or similar bioreactor. A
chemostat allows for the continual addition of fresh medium while
culture liquid is continuously removed to keep the culture volume
constant. By altering the rate at which medium is added to the
chemostat, the growth rate of the microorganism can be
controlled.
[0176] The continuous and/or near-continuous production of a
compound of interesting, such as 1,4-BDO can include culturing a
compound-producing organism in sufficient nutrients and medium to
sustain and/or nearly sustain growth in an exponential phase.
Continuous culture under such conditions can include, for example,
1 day, 2, 3, 4, 5, 6 or 7 days or more. Additionally, continuous
culture can include 1 week, 2, 3, 4 or 5 or more weeks and up to
several months. Alternatively, organisms that produce a compound of
interest can be cultured for hours, if suitable for a particular
application. It is to be understood that the continuous and/or
near-continuous culture conditions also can include all time
intervals in between these exemplary periods. It is further
understood that the time of culturing the compound-producing
microbial organism is for a sufficient period of time to produce a
sufficient amount of product for a desired purpose.
[0177] In some embodiments, the culture can be conducted under
aerobic conditions. An oxygen feed to the culture can be
controlled. Oxygen can be supplied as air, enriched oxygen, pure
oxygen or any combination thereof. Methods of monitoring oxygen
concentration are known in the art. Oxygen can be delivered at a
certain feed rate or can be delivered on demand by measuring the
dissolved oxygen content of the culture and feeding accordingly
with the intention of maintaining a constant dissolved oxygen
content. In other embodiments, the culture can be conducted under
substantially anaerobic conditions. Substantially anaerobic means
that the amount of oxygen is less than about 10% of saturation for
dissolved oxygen in liquid media. Anaerobic conditions include
sealed chambers of liquid or solid medium maintained with an
atmosphere of less than about 1% oxygen.
[0178] Fermentations can be performed under anaerobic conditions.
For example, the culture can be rendered substantially free of
oxygen by first sparging the medium with nitrogen and then sealing
culture vessel (e.g., flasks can be sealed with a septum and
crimp-cap). Microaerobic conditions also can be utilized by
providing a small hole for limited aeration. On a commercial scale,
microaerobic conditions are achieved by sparging a fermentor with
air or oxygen as in the aerobic case, but at a much lower rate and
with tightly controlled agitation.
[0179] In some embodiments, the compound of interest, including
1,4-BDO, can be produced in an anaerobic batch fermentation using
genetically modified E. Coli. In fermentation, a portion of the
feedstock substrate is used for cell growth and additional
substrate is converted to other fermentation byproducts. Media
components such as salts, buffer, nitrogen, etc can be added in
excess to the fermentation to support cell growth. The fermentation
broth is thus a complex mixture of water, the compound of interest,
byproducts, residual media, residual substrate, and feedstock/media
impurities. It is from this fermentation broth that the compound of
interest is isolated and purified. An exemplary fermentation broth
composition is shown below in Table 4.
TABLE-US-00005 TABLE 4* Quantity Component ~100 g/L 1,4-BDO ~5 g/L
cell mass ~10 g/L byproducts (ethanol, acetic acid,
4-hydroxybutyric acid, GBL, proteins) <10 g/L residual
media/salts <1 g/L residual sucrose/glucose <2 g/L
"unfermentables" (feedstock/impurities) *Balance water
[0180] A product concentration of about 5-15% by weight of 1,4-BDO
can be achieved through fermentation based biosynthetic production
processes.
[0181] It is understood that modifications which do not
substantially affect the activity of the various embodiments of
this invention are also included within the definition of the
invention provided herein. Accordingly, the following examples are
intended to illustrate but not limit the present invention.
Example I
Centrifugation of Fermentation Broth
[0182] This example shows the use of a disc-stack centrifuge to
remove cell mass and other solids from a fermentation broth.
[0183] 1,4-Butanediol fermentation broth produced by a genetically
modified E. coli was clarified via centrifugation. A GEA-Westfalia
disc stack centrifuge was used for this step. The lab-scale
centrifuge, model CTC 1 Whispefuge, has a bowl capacity of 1.0
liters and a solids holding space of 0.55 liters. The bowl hood,
distributor, disc stacks, and all process wetted parts are
constructed with high tensile strength stainless steel. The feed to
centrifuge unit was controlled using a peristaltic pump, with the
flow rate held constant at approximately 0.25 liters per minute. A
back pressure of about 15 psi was maintained in the system by
throttling a regulating valve on the outlet centrate flow. The
centrifuge was operated at 12,000 rpm and the feed was at ambient
room temperature. The centrifugation removes cellular biomass and
insoluble materials from the fermentation broth. The concentration
of cellular biomass and insoluble material is indicated by the
turbidity, as measured by optical density (OD) at 600 nm. The
turbidity data for feed fermentation broth and clarified centrate
is shown in Table 5. The feed was visibly hazy and had a measured
OD of 13.3. The clarified centrate was visually much clearer and
had a measured OD of 0.18. Overall the turbidity was decreased by
approximately 99%, showing excellent clarification by the disc
stack centrifuge.
TABLE-US-00006 TABLE 5 Turbidity measured by optical density (OD)
at 600 nm OD at 600 nm Feed, Fermentation Broth 13.3 Clarified
Centrate 0.18
Example II
Ultrafiltration of Fermentation Broth
[0184] This example shows the ultrafiltration of the fermentation
broth following removal of cell mass and other solids by
centrifugation conducted in Example I.
[0185] A GEA lab scale filtration unit, Model L, was used to
further clarify the product produced in Example 1. The Model L
filtration unit was equipped with Hydranautics 5K PES flat sheet
membranes. Total installed membrane area was 0.144 m.sup.2. The
transmembrane pressure was maintained at approximately 36 psi by
adjusting inlet flow and back pressure regulating valve. The
temperature of the feed was maintained at approximately 27.degree.
C. using an inlet heat exchanger. The permeate flow rate was
measured throughout the course of the experiment to determine the
flux. Table 6 shows the permeate flux in liters/m.sup.2/h as a
function of the volumetric concentration factor (VCF).
TABLE-US-00007 TABLE 6 Ultrafiltration flux versus VCF Flux VCF
liters/m.sup.2/h 1.18 15.02 1.44 15.37 1.86 15.29 2.60 15.34 4.33
15.06 6.50 14.79
[0186] Samples were also drawn throughout the experiment to
determine the permeate quality. Protein concentration in the feed
and permeate was measured using Bradford Assay. Table 7 shows the
protein concentration in the feed, permeate and retentate. The
protein concentration decreased by approximately 68% in the
permeate compared to the feed.
TABLE-US-00008 TABLE 7 Protein concentration measured using
Bradford Assay Protein Concentration, mg/L Feed, Centrifuged Broth
84.09 UF Permeate 27.11 UF Retentate 248.90
Example III
Nanofiltration of Fermentation Broth
[0187] This example shows the nanofiltration of the fermentation
broth following ultrafiltration conducted in Example II.
[0188] A GEA lab scale filtration unit, Model L, was equipped with
GE DK nanofiltration flat sheet membranes. Total installed membrane
area was 0.072 m.sup.2. This set up was used to filter the UF
permeate obtained from Example 2. The transmembrane pressure was
maintained at approximately 270 psi by adjusting inlet flow and
back pressure regulating valve. The temperature of the feed was
maintained at 38.degree. C. using an inlet heat exchanger. The
permeate flow was measured throughout the course of the experiment
to determine the flux. Table 8 shows the flux in liters/m.sup.2/h
as a function of the volumetric concentration factor (VCF).
TABLE-US-00009 TABLE 8 Nanofiltration flux Flux VCF liters/m2/h
1.33 14.69 1.74 13.41 2.50 10.42
[0189] Samples were also drawn throughout the experiment to
determine the permeate quality. Organic acids where measured using
LC-MS, salt ions where analyzed using Chromatography (IC), and
glucose was measured using an Analox G6 analyzer. Table 9 shows the
percent rejection for glucose, ions, and organic acids. At the pH
of the feed it is expected that the organic acids are present in
their salt form. The nanofiltration permeate also had a visual
reduction in color from a distinct yellow in the feed to a very
faint yellow in the permeate product.
TABLE-US-00010 TABLE 9 Percent rejection of glucose, ions and
organic acids by nanofiltration Monovalent Divalent Organic Glucose
Cations Cations Anions Acids 88.57% 72.80% 100.00% 82.45%
64.39%
Example IV
Ion Exchange of Fermentation Broth
[0190] This example shows the ion exchange chromatographic
purification of the fermentation broth following nanofiltration
conducted in Example III.
[0191] Nanofiltration permeate obtained from Example III was
processed through an ion exchange step to remove the remaining ions
and further clarify the product. Amberlite IR 120H, a strong acid
cation exchange resin, and Amberlite IRA 67, a weak base anion
exchange resin, were used for this step. Individual cation and
anion exchange columns, 2 ft high.times.1 inch diameter, were
loaded with 5.3.times.10.sup.-3 ft.sup.3 of cation and anion
exchange resins, respectively. The nanofiltration permeate was
first fed to cation exchange column, and then to anion exchange
column at 10 mL/min and 40.degree. C. The ion exchange was analyzed
for ion content via IC. All the remaining ions were removed to a
concentration of less than 0.1 mEq/L. All the remaining organic
acids were also removed in this step. The product was very clear
with no visible yellow color.
Example V
Evaporative Crystallization of a Synthetic Feed
[0192] This example shows the removal of salts from a synthetic
feed by evaporative crystallization on laboratory scale with the
aid of a rotary evaporator.
[0193] Evaporation was performed using a Buchi Rotavap R-205 at
bath temperature of 50.degree. C. and a vacuum of .about.100 mm of
Hg. A synthetic feed material was prepared with about 8% BDO in
water containing approximately 92 mEq/L monovalent cations, 5 mEq/L
divalent cations and 125 mEq/L anions. The water was evaporated off
from this mixture while the salt ions where simultaneously allowed
to precipitate from the solution. Ion concentrations in the
solution were monitored throughout the evaporation by taking small
sample aliquots for analysis by Ion Chromatography. Prior to
analysis the precipitated solids were filtered off. Table 10 shows
the concentration of ions in solution (normalized to 100% in the
feed sample) as the BDO was concentrated from approximately 10 to
95%. The ion concentrations increased up to the saturation point in
the solution (at approximately 30% BDO). After this point further
evaporation forced crystallization (precipitation) of the salts.
Overall, this evaporative crystallization step caused 97.5% of the
salt ions to precipitate from the BDO solution.
TABLE-US-00011 TABLE 10 Evaporative precipitation of synthetic
broth % % Time, BDO Monoatomic Diatomic % h wt % cations cations
Anions 0 10.00 100.00 100.00 100.00 0.25 15.74 159.75 132.32 161.25
0.5 33.79 344.61 159.73 353.02 0.75 81.32 35.49 0.00 47.24 1.5
94.25 22.43 0.00 20.17
Example VI
Salt Solubility
[0194] This example shows salt solubility profiles in various small
carbon chain diols, including 1,4-BDO.
[0195] Salt solubility in different solutions containing varying
amounts of 1,4-Butanediol (BDO), 1,3-Propanediol (PDO) or
1,2-Ethanediol (mono etheylene glycol, MEG) was measured. The salts
were added to 10 mL of the solution until the solution was
saturated. The saturated salt solubility was measured using Ion
Chromatography. Table 11 shows salt solubility of four different
salts at room temperature (approximately 20.degree. C.). The salt
solubility decreases significantly with increases in BDO
concentration demonstrating the feasibility of salt removal using
evaporative crystallization. Table 12 shows salt solubility of
three different salts in different concentrations of 1,4-BDO, PDO
or MEG solutions at room temperature. The results show a decrease
in salt solubilities going from MEG, to PDO, to 1,4-BDO,
demonstrating that 1,4-BDO is best suited for an evaporative
crystallization among the three compounds.
TABLE-US-00012 TABLE 11 Solubility of four different salts in
solutions containing 0 to 100% 1,4-Butanediol (1,4-BDO) Solution
Average Measured Solubility (wt %) ~20 C. (% 1,4-BDO)
KH.sub.2PO.sub.4 NaCl MgSO.sub.4 (NH.sub.4).sub.2SO.sub.4 0 (Water)
20.101 32.300 34.354 57.375 33 3.296 10.803 7.346 11.639 80 0.056
2.529 0.035 0.173 90 0.011 0.314 0.015 0.022 95 0.005 0.314 0.028
0.008 98 0.031 0.175 0.011 0.005 100 0.003 0.050 0.009 0.004
TABLE-US-00013 TABLE 12 Solubility of three different salts in 50,
80 or 100% of 1,4-Butanediol (BDO), 1,3-Propanediol (PDO) or
1,2-Ethanediol (MEG) Solution Average Measured Solubility (wt %)
~20 C. Solvent (% Solvent) KCl Na.sub.2SO.sub.4
(NH.sub.4)H.sub.2PO.sub.4 BDO 50 8.21 0.63 2.97 80 0.94 0.04 0.16
100 0.05 0.00 0.01 PDO 50 8.70 2.38 3.62 80 1.45 0.10 0.46 100 0.30
0.01 0.09 MEG 50 14.11 9.42 8.06 80 7.72 1.81 2.90 100 4.76 0.69
1.22
[0196] Throughout this application various publications have been
referenced within parentheses. The disclosures of these
publications in their entireties are hereby incorporated by
reference in this application in order to more fully describe the
state of the art to which this invention pertains.
[0197] Although the invention has been described with reference to
the disclosed embodiments, those skilled in the art will readily
appreciate that the specific examples and studies detailed above
are only illustrative of the invention. It should be understood
that various modifications can be made without departing from the
spirit of the invention. Accordingly, the invention is limited only
by the following claims.
* * * * *