U.S. patent application number 14/111425 was filed with the patent office on 2014-02-06 for apparatus and method for separating and refining product manufactured by microbial fermentation by using adsorbent.
This patent application is currently assigned to GS CALTEX CORPORATION. The applicant listed for this patent is Jung-Hee Cho, Jin Dal Rae Choi, Moon-Ho Eom, Sang-Jun Jeon, Julia Lee, Sang-Hyun Lee. Invention is credited to Jung-Hee Cho, Jin Dal Rae Choi, Moon-Ho Eom, Sang-Jun Jeon, Julia Lee, Sang-Hyun Lee.
Application Number | 20140038250 14/111425 |
Document ID | / |
Family ID | 47009871 |
Filed Date | 2014-02-06 |
United States Patent
Application |
20140038250 |
Kind Code |
A1 |
Lee; Sang-Hyun ; et
al. |
February 6, 2014 |
APPARATUS AND METHOD FOR SEPARATING AND REFINING PRODUCT
MANUFACTURED BY MICROBIAL FERMENTATION BY USING ADSORBENT
Abstract
The present invention relates to an apparatus and a method for
fermenting, separating, and refining a product, which is produced
by cultivating a microorganism. The apparatus and the method for
fermenting, separating, and refining, of the present invention, can
separate and refine the product that is produced by microbial
fermentation in a simple, continuous manner and with high
efficiency.
Inventors: |
Lee; Sang-Hyun; (Daejeon,
KR) ; Eom; Moon-Ho; (Seoul, KR) ; Lee;
Julia; (Daejeon, KR) ; Jeon; Sang-Jun;
(Daejeon, KR) ; Cho; Jung-Hee; (Seongnam-si,
KR) ; Choi; Jin Dal Rae; (Daejeon, KR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Lee; Sang-Hyun
Eom; Moon-Ho
Lee; Julia
Jeon; Sang-Jun
Cho; Jung-Hee
Choi; Jin Dal Rae |
Daejeon
Seoul
Daejeon
Daejeon
Seongnam-si
Daejeon |
|
KR
KR
KR
KR
KR
KR |
|
|
Assignee: |
GS CALTEX CORPORATION
Seoul
KR
|
Family ID: |
47009871 |
Appl. No.: |
14/111425 |
Filed: |
April 13, 2012 |
PCT Filed: |
April 13, 2012 |
PCT NO: |
PCT/KR2012/002845 |
371 Date: |
October 11, 2013 |
Current U.S.
Class: |
435/136 ;
435/132; 435/148; 435/289.1 |
Current CPC
Class: |
B01D 15/08 20130101;
Y02E 50/10 20130101; C12P 7/16 20130101; C12M 47/10 20130101; C12M
23/58 20130101; C12M 21/12 20130101; C12M 47/12 20130101; C12P 7/06
20130101; B01D 15/1885 20130101; Y02E 50/17 20130101; C12M 25/20
20130101; C12P 7/065 20130101; C12P 7/28 20130101; C12M 25/18
20130101 |
Class at
Publication: |
435/136 ;
435/289.1; 435/132; 435/148 |
International
Class: |
C12M 1/00 20060101
C12M001/00; C12P 7/28 20060101 C12P007/28; C12P 7/06 20060101
C12P007/06; C12P 7/16 20060101 C12P007/16 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 14, 2011 |
KR |
10-2011-0034883 |
Claims
1. A continuous fermentation, separation, and refinement apparatus
of products comprising: a culture bath in which a microbe is
cultured together with a raw material to produce the products; at
least two columns filled with an adsorbent; and a conversion part
controlling so that a culture medium containing the products is
supplied from the culture bath to a specific column, wherein the
conversion part stops supplying the culture medium to a first
column in the case in which the products are sufficiently adsorbed
in the first column supplied with the culture medium and changes a
flow of the culture medium so as to supply the culture medium to a
second column.
2. The continuous fermentation, separation, and refinement
apparatus of claim 1, wherein the case in which the products are
sufficiently adsorbed is a case in which an adsorption rate of the
product with respect to the adsorbent is decreased or a case in
which a concentration of the product in a discharge solution
discharged from the column is 80% or more of a concentration of the
product in the culture medium supplied to the column.
3. The continuous fermentation, separation, and refinement
apparatus of claim 1, wherein at least some of the discharge
solution discharged from the column is supplied to the culture
bath.
4. The continuous fermentation, separation, and refinement
apparatus of claim 3, wherein the case in which the products are
sufficiently adsorbed is a case in which an adsorption rate of the
product with respect to the adsorbent is decreased, a case in which
a concentration of the product in the discharge solution discharged
from the column is 80% or more of a concentration of the product in
the culture medium supplied to the column, a case in which growth
of the microbe in the culture bath is inhibited by an increase in
the concentration of the product, or a case in which productivity
of the product of the microbe is decreased.
5. The continuous fermentation, separation, and refinement
apparatus of claim 1, wherein desorption of the product adsorbed in
the adsorbent proceeds in the first column to which supply of the
culture medium is stopped, while adsorption of the product proceeds
in the second column to which the culture medium is supplied.
6. The continuous fermentation, separation, and refinement
apparatus of claim 5, wherein the desorption is performed using
heat or an eluant.
7. The continuous fermentation, separation, and refinement
apparatus of claim 5, wherein the desorption is performed by
applying heat to the adsorbed product in the adsorbent to vaporize
the product.
8. The continuous fermentation, separation, and refinement
apparatus of claim 5, wherein the desorption is performed using the
eluant, which is an organic solvent, or an acidic or basic aqueous
solution.
9. The continuous fermentation, separation, and refinement
apparatus of claim 1, wherein the product is alcohol, ketone,
ester, or carboxylic acid.
10. The continuous fermentation, separation, and refinement
apparatus of claim 1, wherein the adsorbent is filled at a volume
fraction of 0.1 to 99% based on a volume of the column.
11. The continuous fermentation, separation, and refinement
apparatus of claim 1, wherein a type of the column to which the
culture medium is supplied is a slurry reactor type, a fluidized
reactor type, or a packed reactor type.
12. The continuous fermentation, separation, and refinement
apparatus of claim 1, wherein the raw material is fatty acids or
sugars.
13. The continuous fermentation, separation, and refinement
apparatus of claim 1, further comprising a storage bath storing the
product desorbed from the adsorbent.
14. The continuous fermentation, separation, and refinement
apparatus of claim 1, further comprising a stirrer stirring the
adsorbent and the product in the column.
15. The continuous fermentation, separation, and refinement
apparatus of claim 1, further comprising a filter preventing the
adsorbent from being eluted to thereby be lost.
16. A continuous fermentation, separation, and refinement method of
products comprising: culturing a microbe together with a raw
material to produce the products; supplying a culture medium
containing the products to a first column filled with an adsorbent;
and stopping supplying the culture medium to the first column in
the case in which the products are sufficiently adsorbed in the
first column and supplying the culture medium to a second
column.
17. The continuous fermentation, separation, and refinement method
of claim 16, wherein the case in which the products are
sufficiently adsorbed is a case in which an adsorption rate of the
product with respect to the adsorbent is decreased or a case in
which a concentration of the product in a discharge solution
discharged from the column is 80% or more of a concentration of the
product in the culture medium supplied to the column.
18. The continuous fermentation, separation, and refinement method
of claim 16, wherein the raw material is fatty acids or sugars.
19. The continuous fermentation, separation, and refinement method
of claim 16, wherein the product is alcohol, ketone, ester, or
carboxylic acid.
20. The continuous fermentation, separation, and refinement method
of claim 16, wherein the adsorbent is filled at a volume fraction
of 0.1 to 99% based on a volume of the column.
21. The continuous fermentation, separation, and refinement method
of claim 16, wherein a type of the column to which the culture
medium is supplied is a slurry reactor type, a fluidized reactor
type, or a packed reactor type.
22. The continuous fermentation, separation, and refinement method
of claim 16, wherein at least some of the discharge solution
discharged from the first column is used in culturing the microbe
again.
23. The continuous fermentation, separation, and refinement method
of claim 22, wherein the case in which the products are
sufficiently adsorbed is a case in which an adsorption rate of the
product with respect to the adsorbent is decreased, a case in which
a concentration of the product in the discharge solution discharged
from the column is 80% or more of a concentration of the product in
the culture medium supplied to the column, a case in which growth
of the microbe in the culture bath is inhibited by an increase in
the concentration of the product, or a case in which productivity
of the product of the microbe is decreased.
24. The continuous fermentation, separation, and refinement method
of claim 16, further comprising, desorbing the product adsorbed in
the adsorbent in the first column to which supply of the culture
medium is stopped while adsorption of the product proceeds in the
second column to which the culture medium is supplied.
25. The continuous fermentation, separation, and refinement method
of claim 24, wherein the desorption is performed using heat or an
eluant.
26. The continuous fermentation, separation, and refinement method
of claim 24, wherein the desorption is performed by applying heat
to the adsorbed product in the adsorbent to vaporize the
product.
27. The continuous fermentation, separation, and refinement method
of claim 24, wherein the desorption is performed using the eluant,
which is an organic solvent, or an acidic or basic aqueous
solution.
Description
TECHNICAL FIELD
[0001] The present invention relates to an apparatus and a method
for continuously separating and refining products produced by
microbial fermentation.
BACKGROUND ART
[0002] Butanol can be used as a chemical intermediate in cosmetics,
perfume, hormone, a sanitizer, an industrial coating agent, a paint
additive, fiber, a plastic monomer, medical supplies, vitamins,
antibiotics, pesticides, or the like (Document [Durre, Biotechnol.
J, 2:1525-1534, 2007]).
[0003] As the existing method for preparing butanol, a method of
fermenting sugar using Clostridium strain to produce butanol,
acetone, and ethanol (Document [US Patent Laid-Open Publication No.
1,315,585]) has been used until the 1980s, but thereafter, an oxo
process of synthesizing butanol from propylene obtained from
petroleum has been widely used. However, since the method for
preparing butanol based on petroleum is complicated due to using
high temperature and high pressure and large amounts of hazardous
wastes and carbon dioxide are discharged (Document [Tsuchida et
al., Ind. Eng. Chem. Res., 45: 8634, 2006]). Recently, a demand for
eco-friendly producing butanol from sustainable resources through
microbial fermentation has been increased again.
[0004] However, as described above, currently, in most cases,
butanol has been produced by a chemical synthesis method. An
interest in research into biobutanol has been rapidly increased
around the world due to an increase in oil price, environmental
problems, and the like, but biobutanol has not yet been efficiently
produced.
[0005] In the case of butanol, up to now, in most of the examples
of producing butanol through fermentation, Clostridium strain is
used. There is an example in which productivities of acetone,
butanol, and ethanol were increased by 95%, 37%, and 90%,
respectively, as compared to the case of wild-type strains by
inserting three genes, that is, acetoacetic acid decarboxylase
(adc), CoA transferase A (ctfA), and CoA transferase B (cftB), into
a vector and using a promoter of adc to thereby construct an
artificial operon and then introducing this plasmid pFNK6 into
Clostridium acetobutylicum ATCC 824 strains (Document [Mermelstein
et al., Biotechnol. Bioeng., 42:1053, 1993]). In addition, there is
an example in the recombinant strain cloning and expressing
alcohol/aldehyde dehydrogenase(aad) produced higher amount of
butanol and ethanol than aceton when compared with wild type
(Document [Nair et al., J. Bacteriol., 176:871, 1994]). Otherwise,
as a method for inactivating a function of genes, there is an
example of inactivating butyrate kinase (buk) and
phosphotransacetylase (pta). It was reported that when a strain
PJC4BK in which the buk gene was inactivated was fermented at pH of
5.0 or more, a production amount of butanol was significantly
increased up to 16.7 g/L (Document [Harris et al., Biotechnol.
Bioeng., 67:1, 2000]). However, it was reported that when the case
of a strain in which a pta gene was inactivated was compared with
the case of a wild-type strain, there was no significant difference
in producing a solvent (Document [Harris et al., Biotechnol.
Bioeng., 67:1, 2000]). In addition, it was reported that when
fermentation was performed using a Clostridium beijerinckii BA101
strain, which is a mutant strain induced by random mutation, and
maltodextrin as a carbon source, 18.6 g/L of butanol was produced
(Document [Ezeji et al., Appl. Microbiol. Biotechnol., 63:653,
2004]). However, even in the case of using these recombinant
strains, the production amount of butanol in a culture medium was
significantly low (20 g/L or less) due to toxicity of butanol,
which is a final product, such that it was impossible to
industrially use these recombinant strains. Therefore, various
method for extracting butanol in situ produced during a culture
process to maintain a concentration of butanol in a culture medium
at a level at which cytotoxicity is not generated have been
developed. For example, it was reported that productivity may be
increased by adsorbing butanol produced during continuous culture
using activated carbon (Document [US Patent Registration No.
4520104]).
[0006] However, in this method, only butanol is selectively
adsorbed by the activated carbon and a concentration of the
adsorbed butanol is low, such that it is difficult to recover
butanol, and physical stability of activated carbon is
insufficient, such that it is impossible to reuse the activated
carbon. Therefore, there is a disadvantage in that extraction of
butanol is expensive. An adsorption amount of butanol is in
proportion to a concentration of butanol, but the concentration of
butanol produced in continuous culture is low, such that the
adsorption amount of butanol is also significantly low. Due to this
problem, in spite of the continuous culture process, the
productivity is not over 1 g/L/h. In addition, a column filled with
the activated carbon may be physically clogged due to aggregation
of cells, thereby causing a problem in a process. In this method,
cell aggregates may clog the column and form a channel in a flow of
the culture medium, such that it is difficult to allow the products
such as butanol, acetone, isopropanol, ethanol, or the like, to be
adsorbed in the entire adsorbent, thereby decreasing adsorption
efficiency. A method of using an adsorbent except for activated
carbon to increase productivity and a concentration of a solvent
and using a recyclable adsorbent has been reported (Document
[Nielsen et al., Bioeng. Biotech. 102:811-821, 2009]). The method
is a method of adding the adsorbent in a culture medium and
adsorbing butanol produced during a culture process in the
adsorbent to recover butanol. According to this method, the
adsorbent should be recovered from the culture medium, and
essentially, a loss of the adsorbent is generated in a recovering
process. In addition, impurities produced by microbes in the
culture process and sugar, which is a raw material, are
simultaneously adsorbed, such that purity and productivity of
butanol are low at the time of recovering butanol. Further, an
adsorption amount of butanol is in proportion to an amount of
adsorbent added to the culture medium, but in this method, there is
a limitation in an addition amount of the adsorbent in the culture
medium. In addition, in this method, concentrations of ethanol and
acetone are relatively high, which serves to desorb the adsorbed
butanol, such that there is a limitation in increasing the
concentration of butanol.
DISCLOSURE
Technical Problem
[0007] An object of the present invention is to provide a
separation and refinement apparatus capable of continuously
separating and refining products produced from a fermented culture
medium of a microbe, and a separation and refinement method.
Technical Solution
[0008] In one general aspect, there is provided a continuous
fermentation, separation, and refinement apparatus of products
including:
[0009] a culture bath in which a microbe is cultured together with
a raw material to produce the products;
[0010] at least two columns filled with an adsorbent; and
[0011] a conversion part controlling so that a culture medium
containing the products is supplied from the culture bath to a
specific column,
[0012] wherein the conversion part stops supplying the culture
medium to a first column in the case in which the products are
sufficiently adsorbed in the first column supplied with the culture
medium and changes a flow of the culture medium so as to supply the
culture medium to a second column.
[0013] In another aspect of the present invention, there is
provided a continuous fermentation, separation, and refinement
method of products including:
[0014] culturing a microbe together with a raw material to produce
the products;
[0015] supplying a culture medium containing the products to a
first column filled with an adsorbent; and
[0016] stopping supplying the culture medium to the first column in
the case in which the products are sufficiently adsorbed in the
first column and supplying the culture medium to a second
column.
Advantageous Effects
[0017] With the fermentation, separation, and refinement apparatus
and method according to the present invention, products in the
culture medium of a microbe may be simply and rapidly separated and
purified.
DESCRIPTION OF DRAWINGS
[0018] FIG. 1 is a conceptual diagram showing a fermentation,
separation, and refinement apparatus according to the present
invention.
[0019] FIG. 2 is a comparison graph showing relative butanol
adsorption performance of adsorbents.
[0020] FIG. 3 shows kinetic analysis of butanol adsorption rate to
the adsorbent with various concentrations.
[0021] FIG. 4 shows fermentation profile obtained by performing
culture using a culture method according to the exemplary
embodiment of the present invention (ABE: Acetone, butanol, and
ethanol).
BEST MODE
[0022] Advantages and features of the present invention and methods
to achieve them will be elucidated from exemplary embodiments
described below in detail with reference to the accompanying
drawings. However, the present invention is not limited to the
preferred embodiment disclosed herein but will be implemented in
various forms. The preferred embodiments make disclosure of the
present invention thorough and are provided so that those skilled
in the art can easily understand the scope of the present
invention. Therefore, the present invention will be defined by the
scope of the appended claims. Like reference numerals throughout
the description denote like elements.
[0023] The present invention relates to a continuous fermentation,
separation, and refinement apparatus of products including:
[0024] a culture bath in which a microbe is cultured together with
a raw material to produce the products;
[0025] at least two columns filled with an adsorbent; and
[0026] a conversion part controlling so that a culture medium
containing the products is supplied from the culture bath to a
specific column,
[0027] wherein the conversion part stops supplying the culture
medium to a first column in the case in which the products are
sufficiently adsorbed in the first column supplied with the culture
medium and changes a flow of the culture medium so as to supply the
culture medium to a second column (FIG. 1).
[0028] In addition, the present invention relates to a continuous
fermentation, separation, and refinement method of products
including:
[0029] culturing a microbe together with a raw material to produce
the products;
[0030] supplying a culture medium containing the products to a
first column filled with an adsorbent; and
[0031] stopping supplying the culture medium to the first column in
the case in which the products are sufficiently adsorbed in the
first column and supplying the culture medium to a second
column.
[0032] Further, the present invention relates to a continuous
fermentation, separation, and refinement method of products
including:
[0033] culturing a microbe together with a raw material to produce
the products;
[0034] supplying a culture medium containing the products to a
first column filled with an adsorbent;
[0035] stopping supplying the culture medium to the first column in
the case in which the products are sufficiently adsorbed in the
first column and supplying the culture medium to a second column;
and
[0036] desorbing the adsorbed products from the adsorbent in the
first column to which the culture medium is not supplied while the
products are adsorbed in the second column to which the culture
medium is supplied.
[0037] Hereinafter, the fermentation, separation, and refinement
apparatus of products produced by microbial fermentation according
to the present invention (hereinafter, referred to as the
`fermentation, separation, and refinement apparatus`) and the
fermentation, separation, and refinement method of products
produced through microbial fermentation according to the present
invention (hereinafter, referred to as the `fermentation,
separation, and refinement method`) will be described in detail
with reference to the accompanying drawings.
[0038] The fermentation, separation, and refinement apparatus
according to the present invention includes at least two columns
filled with the adsorbent. The fermentation, separation, and
refinement apparatus according to the present invention includes,
preferably, 2 to 20 columns filled with the adsorbent, more
preferably, 2 to 10 columns, further more preferably, 2 to 5
columns, and most preferably, 2 to 3 columns. These columns may be
represented by a first column, a second column, a third column, a
fourth column, a fifth column, a sixth column, or the like.
However, for convenience of explanation, hereinafter, the present
invention will be described based on two columns. As used herein,
the term "the first and second columns" means arbitrarily numbered
columns for convenience of explanation, but does not mean that the
fermentation, separation, and refinement apparatus includes only
two columns. In addition, according to the present invention, the
culture medium is continuously supplied to several columns, such
that a description of one column is similarly applied to other
columns.
[0039] In a culture bath 130, the microbe is cultured by being
supplied with the raw material and a medium from a supply bath 140.
As a result, in the culture bath 130, the products are produced in
the fermented culture medium (hereinafter, referred to as a
`culture medium`) by microbial fermentation.
[0040] The culture medium discharged from the culture bath 130 is
supplied to a first column 110 using a pump 160. The culture medium
may be continuously or discontinuously supplied. The product
produced by microbe fermentation is contained in the culture medium
supplied to the first column 110, and the product in the culture
medium is adsorbed in an adsorbent 112 in the column.
[0041] In this case, the fermentation, separation, and refinement
apparatus according to the present invention may further include a
stirrer (not shown) stirring the adsorbent and the product in the
column. The culture medium and the adsorbent in the first column
110 are uniformly mixed with each other using the stirrer, thereby
making it possible to prevent the culture medium and the adsorbent
from being aggregated in the column, particularly, at a portion to
which the culture medium is supplied, a portion at which a
discharge solution is discharged outside of the column, or the
like. The culture medium may circulate from an upper portion of the
first column 110 to a lower portion thereof, but is not limited
thereto.
[0042] When the product is sufficiently adsorbed in the first
column 110, connection between the culture bath 130 and the first
column 110 is blocked by a first conversion part 170, such that
supply of the culture medium to the first column 110 is stopped.
Further, the first conversion part 170 and a third conversion part
174 are opened in a direction in which the culture bath 130 and a
second column 120 are connected to each other, such that the
culture medium is supplied to the second column 120. That is, when
the product is sufficiently adsorbed in the first column 110, a
flow of the culture medium is changed so that supply of the culture
medium to the first column 110 is stopped and the culture medium is
supplied to the second column 120. At this time, the first and
third conversion parts 170 and 174 may include a 4-way valve.
[0043] While adsorption is performed in the second column 120 to
which the culture medium is supplied, desorption is performed in
the first column 110 to which supply of the culture medium is
stopped. The term "desorption" means to elute the product adsorbed
in the adsorbent from the adsorbent to allow the adsorbent to be
recyclable.
[0044] The desorption may be performed using heat or an eluant, but
is not limited thereto. In addition, a kind of means used in the
desorption is not limited as long as it is suitable for a process
condition. The desorption may be performed anytime by using heat or
the eluant, and a sufficiently large amount of product may be
separated and purified with a small amount of adsorbent 112.
[0045] For example, desorption in the present invention may be
performed by applying heat to the adsorbed product to vaporize the
product. In this case, in view of cost, the heat generated in a
process is preferable, and the heat may be applied as steam or hot
air. The steam may be water in a vapor state and applied at a
pressure of 0.01 to 6 bar. In addition, the hot air may be applied
at a pressure of 0.01 to 6 bar and have a temperature at which the
product may be eluted in a state in which the column and the
adsorbent are not damaged. For example, the temperature of the hot
air may be 100 to 200.degree. C., preferable 110 to 150.degree. C.,
more preferably 120 to 140.degree. C., and most preferably
130.degree. C. In addition, the desorption in the present invention
may be performed using the eluant, and the eluant may be an organic
solvent, or an acidic or basic aqueous solution. The organic
solvent may be tetrahydrofuran, alcohol, ketone, ether, or ester,
preferably, methanol, acetone, ethylacetate, diethylether, or
methylethylketone, but is not limited thereto.
[0046] As an example of a desorption method, a tetrahydrofuran
solvent is flowed into the column or water vapor is passed through
the column. For example, desorption may be performed by flowing the
tetrahydrofuran solvent having a double volume of a volume of the
adsorbent at a flow rate of 10 mL/min or passing vapor preferably
at 130.degree. C. and 2 bar.
[0047] The desorption proceeds in a state in which the adsorbent in
the first column 110 is not picked out, that is, an in-situ state.
As described above, when the product is adsorbed in-situ in the
adsorbent 112, butanol in the culture medium present in the first
column 110 is maintained at a concentration at which butanol does
not inhibit growth of the microbe or productivity of the product.
While the desorption proceeds in the first column 110, the culture
medium is supplied to the second column 120, such that the culture
medium may be continuously flowed. In addition, in the second
column 120 to which the culture medium is supplied, adsorption
continuously proceeds, such that fermentation, separation, and
refinement of the product may be continuously performed.
[0048] The fermentation, separation, and refinement apparatus
according to the present invention may further include a filter 114
at the upper or lower portion of the column in order to prevent the
adsorbent 112 from being eluted to thereby be lost.
[0049] The case in which the product in the column is sufficiently
adsorbed means a case in which the product is sufficiently adsorbed
in the adsorbent in the column. That is, the product is
sufficiently adsorbed, which means that since the product is
sufficiently adsorbed in the first column, it is judged that the
case of stopping adsorption of the product in the first column and
adsorbing the product in the second column is more preferable as
compared to the case of producing and adsorbing the product in the
first column.
[0050] For example, the case in which the product of the present
invention is sufficiently adsorbed may be the case in which an
adsorption rate of the product with respect to the adsorbent is
decreased or the case in which a concentration of the product in a
discharge solution discharged from the column is 80% or more of a
concentration of the product in the culture medium supplied to the
column. Meanwhile, the product produced by the microbe,
particularly, butanol is contained in the fermented culture medium
of the microbe of the present invention. When the concentration of
butanol in the culture medium reaches at about 12 g/L, the microbe
may be fatally affected by toxicity of butanol. However, at least
some of the discharge solution discharged from the column of the
present invention is supplied to the culture bath, and as the
discharge solution is continuously supplied to the culture bath,
the product in the culture bath, particularly butanol is
accumulated, such that the concentration of butanol is increased,
thereby inhibiting culture of the microbe in the culture bath.
Therefore, the case in which the product of the present invention
is sufficiently adsorbed may be the case in which the growth of the
microbe in the culture bath is inhibited by an increase in the
concentration of the product or the case in which productivity of
the product of the microbe is decreased.
[0051] As a result, the case in which the product is sufficiently
adsorbed may be the case in which the adsorption rate of the
product with respect to the adsorbent is decreased, the case in
which the concentration of the product in the discharge solution
discharged from the column is 80% or more of the concentration of
the product in the culture medium supplied to the column, the case
in which the growth of the microbe in the culture bath is inhibited
by the increase in the concentration of the product, or the case in
which productivity of the product of the microbe is decreased, and
this case may be suitably determined by those skilled in the art
according to the kind of microbe, the kind of adsorbent, the kind
and composition of product, and the like.
[0052] Thereafter, when the product is sufficiently adsorbed in the
second column 120, connection between the culture bath 130 and the
second column 120 is blocked by the first and third conversion
parts 170 and 174, such that supply of the culture medium to the
second column 120 is stopped. Then, the conversion part is opened
in a direction in which the culture bath 130 and a different column
are connected to each other, such that the culture medium is
supplied to the different column. In addition, desorption proceeds
in the second column 120, and the product in the culture medium is
adsorbed in the different column, such that separation and
refinement of the product is continuously performed. In this case,
desorption in the second column 120 proceeds similarly to
desorption in the first column 110.
[0053] The `different column` may be the first column 110 or a
third column. The different column may be changed according to the
number of columns included in the fermentation, separation, and
refinement apparatus. That is, in the case in which the number of
columns in the fermentation, separation, and refinement apparatus
is 3 or more, the `different column` is a third column. In the case
in which the number of columns in the fermentation, separation, and
refinement apparatus is 2, the `different column` is the first
column 110, and the first column 110 is in a state in which
desorption is completed while the adsorption of the product in the
second column 120 proceeds. In this case, the first conversion part
170 is opened in the direction in which the culture bath 130 and
the first column 110 are connected to each other, such that the
culture medium is supplied to the first column 110. In addition,
while desorption proceeds in the second column 120, adsorption of
the product proceeds in the first column 110. The fermentation,
separation, and refinement apparatus according to the present
invention may reuse the adsorbent in the columns by repeating the
above-mentioned process and continuously ferment, separate, and
purify the product.
[0054] Meanwhile, the fermentation, separation, and refinement
apparatus according to the present invention may further include a
storage bath 150 storing the product desorbed from the adsorbent
filled in the first column 110. While desorption proceeds in the
first column 110, second and fourth conversion parts 172 and 176
are opened in a direction in which the storage bath 150 and the
first column 110 are connected to each other. Therefore, the
product desorbed from the first column 110 moves to the storage
bath 150. In this case, the second and fourth conversion parts 172
and 176 may include a 4-way valve.
[0055] In the case in which desorption proceeds in the second
column 120, the desorbed product may also be stored in the storage
bath 150. In this case, the fourth conversion part 176 is opened in
a direction in which the storage bath 150 and the second column 120
are connected to each other, thereby making it possible to move the
desorbed product to the storage bath 150.
[0056] Meanwhile, at least some of the discharge solution
discharged from the column of the present invention may be supplied
to the culture bath. In this case, an amount of discharge solution
supplied to the culture bath may be equal to or less than an amount
of culture medium supplied to the column.
[0057] Hereinabove, the fermentation, separation, and refinement
apparatus and method of the present invention are briefly
described. Hereinafter, the present invention will be described in
detail.
[0058] The fermentation, separation, and refinement apparatus and
method according to the present invention may simply and
continuously ferment, separate, and purify the product produced by
culturing the microbe, and since desorption may be suitably
performed when the product is sufficiently adsorbed in the
adsorbent, adsorption efficiency may also be high. Therefore, the
fermentation, separation, and refinement apparatus and method
according to the present invention may separate and purify the
product produced by culturing the microbe with the high
efficiency.
[0059] As the microbe of the present invention, any microbe may be
used as long as the microbe may produce the product, which is a
biofuel, from the raw material, but the present invention is not
particularly limited. For example, the microbe of the present
invention may be bacteria, yeast, fungus, or the like, and
preferably, bacteria or yeast. The microbe of the present invention
may be a wild-type microbe or genetically modified microbe.
Preferably, the microbe of the present invention may be a wild type
strain of Clostridium genus, E. coli, or the like, or a strain of
which genes are recombined so as to increase productivity of a
specific product, but it is obvious to those skilled in the art
that the present invention is not limited thereto.
[0060] For example, in the case of using bacteria in the
Clostridium genus of which buk gene coding butyrate kinase is
deleted as the microbe of the present invention, there is an
advantage in that products such as butanol, acetone, isopropanol,
ethanol, or the like, may be produced at a high concentration under
anaerobic conditions while not producing a large amount of butyric
acid. For example, in the case of using bacteria in the Clostridium
genus of which buk gene coding butyrate kinase, pta gene coding
phosphotransacetylase, and ackA gene coding acetate kinase are
deleted as the microbe of the present invention, there is an
advantage in that products such as butanol, acetone, isopropanol,
ethanol, or the like, may be produced at a high concentration under
anaerobic conditions while not producing large amounts of other
organic acids. In addition, in the case of using bacteria in the
Clostridium genus of which adc gene coding acetoacetic acid
decarboxylase is deleted as the microbe of the present invention,
there is an advantage in that butanol may be selectively produced
at a high concentration while not producing acetone. Further, in
the case of using bacteria in the Clostridium genus transformed
with a plasmid including genes coding secondary alcohol
dehydrogenase (ald), which is an enzyme converting acetone into
isopropylalcohol, there is an advantage in that high value-added
products such as butanol, isopropanol, ethanol, or the like, may be
produced.
[0061] The gene modified Clostridium may be prepared by a method
disclosed in the released paper (Document [Microbiology (1996),
142, 2079-2086]). For example, PJC4BK or BKM19(KCTC 10558BP), which
is a mutant strain, may be prepared by deleting the butyrate kinase
gene (buk) from Clostridium acetobutylicum. Thereafter, mutant
strains may be prepared by deleting the phosphotransacetylase gene
(pta) and the acetate kinase gene (ackA) from the strain,
respectively. Next, it may be confirmed that when these strains are
cultured under anaerobic conditions, the products such as butanol,
acetone, isopropanol, ethanol, or the like, are produced at a high
concentration.
[0062] The microbe of the present invention may be cultured by a
fed-batch culture method or continuous culture method. The
continuous culture method is a method of supplying a fresh medium
to a culture bath at a predetermined rate and simultaneously
discharging the same amount of fermented culture medium of the
microbe to thereby always maintain the culture medium to be
constant in the culture bath. Meanwhile, the fed-batch culture
method, which is a culture method of intermittently supplying a
medium, is a method of optionally controlling a concentration of a
substrate in the culture medium.
[0063] The raw material of the present invention may be a material
capable of being used by the microbe at the time of fermentation to
thereby produce the product, and a kind thereof is not particularly
limited. For example, the raw material of the present invention may
be biomass, sugars, fatty acids, or the like. The biomass may be a
woody biomass, grains, or the like, but is not limited thereto. The
sugars may be monosaccharides, polysaccharides, polysaccharides, or
the like, be (C3-C12) sugar, and include polysaccharides produced
during a hydrolysis process of the biomass. For example, the sugars
may be glycerol, glucose, sucrose, xylose, starch, cellulose, or
the like, but is not limited thereto. The fatty acid may be
(C2-C24) fatty acid, for example, acetic acid, butyric acid,
propionic acid, or the like, but is not limited thereto.
[0064] The product of the present invention is a fermented product
produced by the microbe. Preferably, the product of the present
invention may be alcohol, ketone, ester, carboxylic acid, or the
like, as a biofuel, but is not limited thereto. For example, the
alcohol may be (C2-C6)alcohol, preferably, alcohol having at most 4
carbon atoms, and more preferably, ethanol, propanol, isopropanol
(2-propanol), 1,2-propanediol, 1,3-propanediol, 1,3-butanediol,
1,4-butanediol, butanol, or the like, but is not limited thereto.
The ketone may be (C3-C8)ketone. For example, the ketone may be
acetoacetate, acetone, or the like, but is not limited thereto. The
ester may be (C4-C8)ester, for example, ethyl acetate, ethyl
butyrate, butyl acetate, butyl butyrate, or the like, but is not
limited thereto. The carboxylic acid may be (C2-C8)carboxylic acid,
for example, acetic acid, butyric acid, propionic acid, or the
like, but is not limited thereto. Preferably, the product of the
present invention may be butanol, isopropanol, ethanol, or acetone,
but is not limited thereto.
[0065] The adsorbent 112 of the present invention may be filled at
a volume fraction of 0.1 to 99% based on a capacity, that is, a
volume of the column containing the adsorbent 112. In the case in
which the volume fraction of the adsorbent is less than 0.1 volume
% based on the volume of the column, the amount of the adsorbent
112 is insufficient, such that adsorption may not be suitably
performed. Further, in the case in which the volume fraction of the
adsorbent is 99 volume % or more, aggregates of the adsorbent 112
or cells are formed in the column, such that adsorption may not be
suitably performed.
[0066] A type of column to which the culture medium is supplied may
be a slurry reactor type, a fluidized reactor type, or a packed
reactor type. The fluidized reactor type column is a column
containing a small amount of an adsorbent and having high fluidity,
the slurry reactor type column is a column containing an adsorbent
suspended in a culture medium, and the packed reactor type column
is a column in which an adsorbent is substantially packed. In this
case, the middle type columns of the three type columns may be
divided into the three type columns depending on which column they
are close to, and although the inside of the column is the middle
type of the three types, the column may be included in any one of
the three types. For example, according to the degree of fluidity,
when it is judged that the adsorbent is packed so as not to
substantially flow, the column may be classified as the packed type
column, and when it is judged that the adsorbent may be suspended,
the column may be classified as the slurry reactor type column.
[0067] The conversion part of the present invention serves to
change and control the flow of the culture medium or discharge
solution and may control so that the culture medium is supplied
from the culture bath 130 to a specific column or send the
discharge solution discharged from a specific column to the storage
bath 150. A single or a plurality of conversion parts may be used,
and the conversion part may be suitably installed by those skilled
in the art according to the design of the fermentation, separation,
and refinement apparatus of the present invention.
Experimental Example 1
[0068] In order to select a suitable adsorbent used in the Example
of the present invention, butanol adsorption performance of various
adsorbents prepared by Mitsubishi Corp. was compared. After various
kinds of adsorbents were added to 50 mL of a phosphate buffer
solution (50 mM) containing 2% butanol and left for 1 hour while
being stirred, a concentration of butanol remaining in the solution
was analyzed using gas chromatography. As a result, it was analyzed
that the adsorbent SP850 had most excellent performance (FIG. 2),
but the kind of adsorbent is not limited to SP850. Analysis of
products such as butanol, acetone, isopropanol, ethanol, or the
like, was performed using the gas chromatography (Agilent, USA),
and analysis conditions were as shown in the following Table 1.
TABLE-US-00001 TABLE 1 Gas chromatography analysis condition
Injector temperature 320.degree. C. detector temperature
320.degree. C. Injector split ratio 20/1 Injection volume 0.1 .mu.L
Oven conditions 80.degree. C./20 min Air flux 300 mL/min H2 flux 30
mL/min Column: Supelco CarboWAX
Experimental Example 2
[0069] In order to confirm an adsorption rate of butanol adsorbed
in the adsorbent SP850, kinetic analysis was performed on
adsorption of butanol. In the culturing used in Example of the
present invention, since butanol should be adsorbed for a short
time for which the culture medium passed through the column, the
adsorption rate of butanol was significantly important. First, 3 g
(dried weight) of the adsorbent was added to 50 mL of liquid
Clostridium growth media (CGM) in which butanol was contained at
various concentrations, and sampling was performed at sample times
of 30 seconds, 1 minute, 2 minutes, 5 minutes, 10 minutes, 30
minutes, and 1 hour while stirring the mixture at 200 rpm. Then, a
concentration of butanol remaining in the solution was confirmed
through gas chromatography analysis.
[0070] As a result, it may be confirmed that butanol was adsorbed
within about 1 minute in various concentration ranges (FIG. 3).
Experimental Example 3
[0071] In order to confirm an amount of adsorbent suitable of
fed-batch fermentation and fermentation conditions, a recombinant
Clostridium strain was constructed. A recombinant Clostridium
strain (C. acetobutylicum PJC4BK-IPA2) containing secondary alcohol
dehydrogenase required for fermenting the microbe was constructed.
The Clostridium strain was cultured in 60 mL of liquid Clostridium
growth media (CGM, 0.75 g/L K2HPO4, 0.75 g/L KH2PO4, 0.7 g/L,
MgSO4H2O, 0.017 g/L MnSO4H2O, 0.01 g/L, FeSO4H2O, 2 g/L (NH4)2SO4,
1 g/L NaCl, 2 g/L asparagine, 0.004 g/L p-aminobenzoic acid, 5 g/L
yeast extract, 4.08 g/L CH3COONaH2O, and 80 g/L glucose) under
anaerobic conditions until OD600 reached 0.5. Then, the culture
medium was left in ice water for 10 minutes, and the culture medium
was centrifuged with 7000 G at 4.degree. C. for 10 minutes. 15 mL
of sucrose (270 mM) and 0.11 mL of NaH2PO4 (686 mM, pH 7.4) were
mixed with each other, thereby preparing an electroporation buffer
solution. After a cell pellet was washed with the electroporation
buffer solution prepared as described above three times, the washed
cell pellet was suspended in 2 mL of the same buffer solution,
thereby constructing recombinant cells. 0.5 to 2.0 g of plasmid
containing secondary alcohol dehydrogenase gene was added to the
prepared cells (5001) for transformation, and then electroporation
(4 mm cuvette, 2.5 kV, .infin..OMEGA., 25 uF) was performed using
Gene pulser II (Bio-Rad Corp.), followed by anaerobic culture in
the culture medium to which antibiotic was added, thereby
completing the construction of recombinant strain. All of the
plasmids used for transformation were constructed so as not to be
affected by restriction system of the Clostridium strain by being
methylated in E. coli ER2275 transformed with a pAN1 vector before
electroporation.
[0072] The Clostridium strain PJC4BK-IPA2 cultured as described
above was smeared on a CGM/chlorampenicol plate medium and
anaerobically cultured overnight at 37.degree. C. 2 cultured
colonies were inoculated in a 50 mL disposable tube in which 20 mL
of CGM/chlorampenicol culture medium was contained and
anaerobically cultured while being placed at 37.degree. C. until
OD600 reached 3. The cultured broth was inoculated again in a
liquid CGM containing 100 mL of 8% glucose and anaerobically
cultured while being placed at 37.degree. C. until OD600 reached 2
to 3. Then, the resultant was inoculated in a culture bath in which
1 L of the liquid CGM and 0 mL (0 g/L, dried weight), 200 mL (60
g/L), 250 mL (75 g/L), 300 mL (90 g/L), 350 mL (105 g/L), and 400
mL (120 g/L) of the adsorbent were contained, respectively, and
cultured. After starting the culture, concentrations of the
produced butanol, acetone, isopropanol, ethanol, and the like, were
analyzed per 3 hours while maintaining a concentration of glucose
at 20 g/L or more. Analysis of products such as butanol, acetone,
isopropanol, ethanol, and the like, was performed using the gas
chromatography (Agilent, USA), and analysis conditions were the
same as in Table 1.
[0073] Concentrations of sugar and organic acid may be confirmed
using high pressure liquid chromatography (HPLC), gas
chromatography, and a sugar analyzer after centrifuging the culture
medium and obtaining a supernatant. Conditions of the HPLC were as
follows: water containing 0.01N sulfuric acid was used as a mobile
phase, and a flow rate was 0.6 mL/min. As the column, Aminex87H and
Aminex87P (Bio-rad, USA) were used, and the produced sugar and
organic acid were analyzed using a reflective index (RI)
detector.
[0074] As a result, it may be confirmed that the recombinant and
mutant strains cultured as described above produced larger amounts
of products such as butanol, acetone, isopropanol, ethanol, and the
like in the culture medium containing the adsorbent, and the
optimal amount of the adsorbent was 250 mL/L, as shown in Table
2.
TABLE-US-00002 TABLE 2 Consumption Amount of Production amount of
adsorbent product (g/L) Yield rate glucose (mL/L) Acetone Ethanol
Butanol IPA Sum (%) (g/L/h) (g/L) 0 0.4 4.1 16.8 3.3 25 35 0.6 70
200 0.6 7.7 23.1 9.6 40 33 1.35 120 250 3.6 8.4 27.6 6.2 46 34 1.2
132 300 2.3 6.2 27.1 7.8 43.3 30 1.09 153 350 7.0 7.2 25.1 5.4 44.7
28 1.06 161 400 6.4 8.53 27.2 7.6 49.7 30 1.10 166
Experimental Example 4
[0075] The same culture medium, culture method, and analysis method
as in the Experimental Examples were used, and 200 mL of the
adsorbent was used, such that reusability of the adsorbent was
tested. After completing the culture, the products such as butanol,
acetone, isobutanol, or ethanol, adsorbed in the adsorbent were
recovered using a column for desorption and eluted by flowing a
tetrahydrofuran solvent having a double volume of a volume of the
adsorbent at a flow rate of 10 mL/min. Then, the total amount of
products was analyzed using gas chromatography, and the total
amount of adsorbed solvent was compared thereto every time, thereby
analyzing performance of the adsorbent.
[0076] As a result, it was confirmed that even though the adsorbent
was reused, adsorption of the product was suitably performed (Table
3).
TABLE-US-00003 TABLE 3 Consumption The amount number Production
Culture of of Product (g/L) Yield rate time glucose reuse Acetone
Ethanol Butanol IPA Sum (%) (g/L/h) (h) (g/L) 1 2.2 6.6 26.9 6.6 42
36 1.02 41 117 2 1.8 6.4 20.0 7.7 36 35 1.20 30 104 3 0.6 7.7 23.1
9.6 40 33 1.35 30 120 4 1.8 7.1 23.2 7.6 40 33 1.32 30 121 5 1.7
4.7 23.4 6.5 37 34 1.23 30 110 6 1.5 7.3 22.9 9.0 41 37 1.03 40 110
7 1.4 6.9 22.3 8.5 39 33 1.02 39 118 8 3.3 5.6 21.4 5.5 36 32 1.00
36 110 9 1.5 6.7 20.9 7.1 36 32 1.01 36 112 10 2.1 6.3 22.9 6.5 38
36 1.26 36 104
Experimental Example 5
Preparation of Products Such as Butanol, Acetone, Isopropanol,
Ethanol, and the Like, Using Fed-Batch Culture Method
[0077] Based on the results optimized in the Experimental Examples,
products such as butanol, acetone, isopropanol, ethanol, and the
like, were prepared using a fed-batch culture method. Experimental
methods and analysis were performed similarly to those in
<Example 3>, but fermentation of glucose contained in the
culture medium was performed in a state in which the concentration
of the CGM culture medium containing 250 mL of the adsorbent was
the same or increased 2 times as compared to <Experimental
Example 3>, respectively, such that amounts of the prepared
products such as butanol, acetone, isopropanol, ethanol, and the
like, were compared with each other.
[0078] As a result, it may be confirmed that the recombinant and
mutant strains cultured as described above produced larger amounts
of butanol, acetone, isopropanol, ethanol, and the like, during the
culture process, depending on an amount of a nutrient, as shown in
Table 4. This result means that in the case of simultaneously
injecting the nutrient and carbon source according to the culture
method in Example of the present invention, the high concentration
products such as butanol, acetone, isopropanol, ethanol, and the
like, may be synthesized with a high yield in the continuous
culture as well as the fed-batch culture.
TABLE-US-00004 TABLE 4 Culture condition Concentration Solvent
Production of Product (g/L) increase Yield rate adsorbent CGM
Acetone Ethanol Butanol IPA Sum rate (%) (%) (g/L/h) .largecircle.
2 0.77 9.70 32.4 9.06 52 108 33 1.24 .largecircle. 1 3.63 8.4 27.6
6.2 46 84 35 1.21 X 1 0.43 4.1 16.8 3.3 25 0 35 0.6
Experimental Example 6
Manufacturing of Fed-Batch Culture Apparatus and Preparation of
Products Such as Butanol, Acetone, Ethanol, and the Like, Using
Culture Method
[0079] The fed-batch culture apparatus shown in FIG. 1 was
manufactured. In this case, two columns were included therein. In
order to prevent the adsorbent from being eluted at upper and lower
portions of the column having a volume of 500 mL to thereby be
lost, a filter (about 150 m) was mounted, and then a stirrer was
mounted. Thereafter, 300 mL of adsorbent SP850 was filled in two
columns (referred to as first and second columns, respectively).
These columns were connected to a culture bath using a silicon
tube, and a pump was mounted so as to circulate a culture medium in
the column. 4-way valves were mounted at an inlet and an outlet of
the column so as to flow an eluant when the products such as
butanol, acetone, ethanol, and the like were sufficiently adsorbed
in the adsorbent in the column during the culture process to
thereby perform desorption in real time. When the product was
sufficiently adsorbed in the first column, the flow of the culture
medium and the product were changed to the second column by
adjusting the 4-way value. In the first column in which the flow of
the culture medium and the product was blocked, desorption
proceeded in a state in which the adsorbent in the first column was
not picked out. While desorption proceeded in the first column,
adsorption continuously proceeded in the second column to which the
culture medium and the product were supplied. Similarly, when the
product was sufficiently adsorbed in the second column, the flow of
the culture medium and the product was changed to the first column
in which desorption was completed by adjusting the 4-way value. The
adsorbent in the first and second columns may be reused by
repeating the above-mentioned processes. A circulation direction of
the culture medium is a direction from the upper portion of the
column to the lower portion thereof, but the circulation direction
did not matter.
[0080] Meanwhile, C. acetobutylicum PJC4BK strains capable of
producing butanol, acetone, ethanol, and the like, were prepared
using a fed-batch culture apparatus and cultured as in the
Experimental Examples. First, 300 mL of seed anaerobically cultured
in the liquid CGM overnight was inoculated and cultured into a
reactor in which 2.7 L of the liquid CGM was contained. In
Experimental Examples of the present invention, the seed was
cultured by general batch fermentation, but in order to prepare the
high concentration products such as butanol, acetone, ethanol, and
the like at a higher cell concentration, cell immobilization type
culture may be performed. The culture medium passed through the
first column via the pump at a flow rate of 30 mL/min
simultaneously with starting the culture. It was confirmed that the
adsorbent SP850 was suspended in the culture medium while the
culture medium passed through the first column to form a slurry
phase, such that the flow of the culture medium was not blocked by
the aggregates of cells, and the culture medium passed through the
first column. In addition, immediately before and after the culture
medium passed through the first column, culture medium samples were
taken, and concentrations of butanol, acetone, ethanol, and the
like, were analyzed using gas chromatography.
[0081] During the culture process, a concentration of sugar was
maintained at 20 g/L using HPLC and a sugar analyzer. Amounts of
the products such as butanol, acetone, isopropanol, ethanol, or the
like were confirmed through gas chromatography.
[0082] As a result, it was confirmed that the fed-batch culture was
stably performed for about 150 hours, and the concentration of the
product in the discharge solution (after adsorption) discharged
from the column was significantly decreased as compared to the
concentration of the product in the culture medium (before
adsorption) supplied to the column. That is, it may be confirmed
that the products such as butanol, acetone, ethanol, and the like,
was maintained at a significantly low concentration in the
discharge solution discharged from the column. In addition, it was
confirmed that the concentration of the product was constantly
maintained in the culture apparatus, such that the microbe was not
affected by the toxicity of butanol but may stably prepare the
product. Further, the products such as butanol, acetone, ethanol,
and the like, adsorbed in the first column were desorbed using
water (95.degree. C.), and as a result, it was confirmed that the
products such as butanol, acetone, ethanol, and the like, were
excellently adsorbed and desorbed. Furthermore, it was confirmed
that even in the case of repeating replacement of the column 20
times or more, the adsorption performance of the column was not
decreased (Tables 5 and 6 and FIG. 4).
TABLE-US-00005 TABLE 5 Consumption Production Culture amount of
Product(g) Yield rate time glucose Acetone Ethanol Butanol Sum (%)
(g/L/h) (hour) (g) Culture medium 14.1 49.9 61.8 125.8 31 1.24 146
2610 Desorption 58.4 106.5 525.7 690.6 Sum 72.4 156.4 587.6
816.4
[0083] Results of preparing acetone, ethanol, and butanol
TABLE-US-00006 TABLE 6 After desorption Ace- Acetone Ethanol
Butanol A tone Ethanol Butanol C D C D C D B* 22 1.1 0.0 5.8 0.6
3.6 1.1 2.1 0.3 8.1 1a 25 2.5 0.9 5.9 1.1 3.6 1.6 3.0 1.6 8.0 1b 29
2.4 1.3 7.7 1.0 3.7 1.8 3.9 2.1 9.1 2a 32 1.7 1.1 8.2 1.3 4.0 2.5
5.0 2.9 10.7 2b 35 1.7 1.3 10.1 1.6 3.8 3.6 5.4 3.8 10.8 3a 41 3.4
3.5 13.9 1.2 3.6 2.6 5.9 4.0 10.6 3b 44 0.0 0.9 5.5 0.5 3.0 2.5 5.6
0.7 9.6 4a 47 1.5 1.7 12.4 0.2 2.8 1.3 5.8 0.6 7.0 4b 50 1.4 1.6
11.2 0.5 2.6 2.5 5.8 0.6 6.4 5a 53 1.5 1.9 10.4 0.6 2.9 2.3 6.2 0.8
6.0 5b 56 1.4 1.8 8.2 0.4 2.8 2.4 6.6 0.5 6.7 6a 59 1.4 2.0 8.2 0.5
2.7 3.0 7.0 0.6 7.0 6b 62 1.1 1.8 8.2 0.8 2.7 4.5 7.7 1.1 8.1 7a 65
0.2 1.1 7.7 0.5 2.6 3.4 8.2 0.5 8.2 7b 68 1.4 2.6 9.0 0.5 2.6 3.9
8.7 0.8 9.0 8a 71 1.2 2.2 12.6 0.5 2.5 3.5 8.6 0.7 8.4 8b 74 1.3
2.1 11.6 0.2 2.5 2.3 8.7 0.3 8.4 9a 77 0.0 1.3 11.8 0.5 2.4 3.9 8.9
1.0 8.7 9b 80 0.9 1.9 11.9 0.4 2.4 3.8 8.9 0.8 8.7 10a 83 0.7 1.7
12.2 0.6 2.4 4.5 9.1 1.0 8.7 10b 86 0.8 1.8 11.7 0.4 2.4 3.8 9.0
0.8 9.1 11a 89 1.0 2.2 13.2 0.5 2.5 4.3 10.0 0.8 10.3 11b 92 1.1
3.1 10.1 0.9 2.3 6.4 10.2 1.7 10.6 12a 94 0.6 1.9 11.6 0.4 2.0 4.2
9.9 0.6 9.1 12b 96 1.2 3.0 13.1 0.6 1.7 5.9 9.4 1.2 7.3 13a 98 1.1
2.9 12.3 0.3 1.6 4.0 9.1 0.6 6.2 13b 101 1.1 3.1 13.6 0.3 1.5 4.0
8.9 0.9 5.8 14a 104 1.0 2.8 12.6 0.3 1.6 3.6 8.7 0.5 8.7 14b 107
1.3 2.9 9.9 0.2 2.1 2.3 8.9 0.3 7.2 15a 109 1.6 3.2 10.2 0.2 2.1
2.7 8.7 0.3 6.7 15b 111 1.7 3.7 8.3 0.3 2.2 2.9 8.6 0.7 7.7 16a 114
1.6 3.1 12.7 0.3 2.2 3.2 8.7 0.8 8.9 16b 116 1.3 2.7 11.6 0.3 2.0
2.7 9.4 0.8 9.0 17a 118 0.7 1.7 11.7 0.4 1.9 3.6 8.4 0.9 7.8 17b
120 1.2 2.9 11.3 0.2 1.9 2.1 9.1 0.8 8.3 18a 122 1.2 2.7 13.2 0.6
1.7 5.0 8.1 1.3 6.7 18b 124 1.2 2.9 10.1 0.3 1.7 3.2 8.4 0.8 7.2
19a 126 1.1 2.7 12.2 0.3 1.7 3.4 8.2 0.7 6.8 19b 128 1.2 2.9 11.2
0.1 1.8 1.5 8.3 0.4 6.9 20a 131 1.2 2.6 13.9 0.2 2.0 2.5 8.8 0.4
8.0 20b 134 0.9 2.1 13.7 0.3 2.2 2.7 9.0 0.6 8.7 21a 136 1.4 2.9
15.5 0.4 2.1 3.5 8.8 0.7 8.4 21b 138 1.2 2.7 12.8 0.3 2.0 3.0 8.6
0.9 7.9 22a 140 1.3 2.9 14.5 0.6 2.0 4.7 8.5 1.1 7.6 22b 142 1.2
3.0 13.4 0.3 2.2 3.0 9.2 0.8 8.1 23a 144 1.3 2.9 15.2 0.3 2.2 3.2
9.3 0.7 8.1 23b 146 1.1 2.3 13.5 0.3 2.2 3.0 9.0 0.8 7.6 24a *a and
b mean the first and second columns, respectively. Unit: g/L A:
Culture time B: The number of replaced column C: After adsorption
D: Before adsorption
DETAILED DESCRIPTION OF MAIN ELEMENTS
[0084] 100: fermentation, separation, and refinement apparatus
[0085] 110: first column 120: second column [0086] 112: adsorbent
114: filter [0087] 130: culture bath 140: supply bath [0088] 150:
storage bath 160: pump [0089] 170: first conversion part 172:
second conversion part [0090] 174: third conversion part 176:
fourth conversion part
* * * * *