U.S. patent application number 17/806219 was filed with the patent office on 2022-09-29 for process and system for product recovery and cell recycle.
The applicant listed for this patent is LanzaTech, Inc.. Invention is credited to Nicholas Bourdakos, Jason Carl Bromley, Robert John Conrado, Allan Haiming Gao, Michael Emerson Martin, Christophe Daniel Mihalcea, Ignasi Palou-Rivera, Paul Alvin Sechrist, Joseph Henry Tizard.
Application Number | 20220305399 17/806219 |
Document ID | / |
Family ID | 1000006394555 |
Filed Date | 2022-09-29 |
United States Patent
Application |
20220305399 |
Kind Code |
A1 |
Sechrist; Paul Alvin ; et
al. |
September 29, 2022 |
PROCESS AND SYSTEM FOR PRODUCT RECOVERY AND CELL RECYCLE
Abstract
The invention is directed to a method for recovering at least
one product from a fermentation broth. The invention relates to the
use of a vacuum distillation vessel to recover products, such as
ethanol, from a fermentation broth, where the fermentation broth
comprises viable microbial biomass, and where the recovery of the
product is completed in such a manner to ensure the viability of
the microbial biomass. The invention provides for product recovery
at an effective rate so as to prevent the accumulation of product
in the fermentation broth. To ensure the viability of the microbial
biomass, the invention is designed to reduce the amount of stress
on the microbial biomass. By ensuring the viability of the
microbial biomass, the microbial biomass may be recycled and reused
in the fermentation process, which may result in an increased
efficiency of the fermentation process.
Inventors: |
Sechrist; Paul Alvin; (South
Barrington, IL) ; Bourdakos; Nicholas; (Toronto,
CA) ; Conrado; Robert John; (Washington, DC) ;
Gao; Allan Haiming; (West Chester, PA) ; Bromley;
Jason Carl; (Chicago, IL) ; Mihalcea; Christophe
Daniel; (Chicago, IL) ; Martin; Michael Emerson;
(Chicago, IL) ; Palou-Rivera; Ignasi; (Flossmoor,
IL) ; Tizard; Joseph Henry; (Skokie, IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
LanzaTech, Inc. |
Skokie |
IL |
US |
|
|
Family ID: |
1000006394555 |
Appl. No.: |
17/806219 |
Filed: |
June 9, 2022 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
16802057 |
Feb 26, 2020 |
|
|
|
17806219 |
|
|
|
|
15926851 |
Mar 20, 2018 |
10610802 |
|
|
16802057 |
|
|
|
|
62473850 |
Mar 20, 2017 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12M 47/10 20130101;
B01D 3/002 20130101; C12N 1/02 20130101; C12M 47/02 20130101; B01D
19/0057 20130101; B01D 3/10 20130101; C12P 7/04 20130101; B01D
53/14 20130101 |
International
Class: |
B01D 3/10 20060101
B01D003/10; C12N 1/02 20060101 C12N001/02; C12M 1/00 20060101
C12M001/00; B01D 3/00 20060101 B01D003/00; B01D 19/00 20060101
B01D019/00 |
Claims
1. A process for removing a product from a fermentation broth, the
process comprising: a. flowing a fermentation broth comprising
microbial biomass and product from a bioreactor to a broth stripper
comprising a top, a bottom; b. partially vaporizing the
fermentation broth in the broth stripper to produce product rich
vapor stream, and product depleted liquid stream comprising live
microbial biomass; c. passing the product depleted liquid stream
back to the bioreactor and passing the product rich vapor stream to
a trim condenser to generate a condensed product stream; d. passing
the condensed product stream to a rectification column comprising a
top and a bottom, to separate a rectification column overhead
stream comprising product and a rectification column bottoms
stream; and e. passing the rectification column bottoms stream to
the bottom of the broth stripper and directly transferring heat
from the bottom of the rectification column to the bottom of the
broth stripper.
2. The process of claim 1 wherein the condensing of the product
vapor stream is incomplete and a secondary condenser is used to
condense remaining vapor.
3. The process of claim 2 wherein the secondary condenser provides
cooling through indirect contact with cooling water or chilled
water.
4. The process of claim 1 further comprising transferring heat,
indirectly, by heat exchanging the rectification column bottoms
stream with the condensed product stream.
5. The process of claim 1, wherein the fermentation broth is
partially vaporised in the broth stripper at a temperature between
37.degree. C. and 50.degree. C. and a pressure between 40 mbar and
100 mbar.
6. The process of claim 1, wherein a viability of the microbial
biomass in the product depleted liquid stream is greater than
85%.
7. The process of claim 1, further comprising storing the product
depleted liquid stream from the broth stripper in a cooled tank
prior to passing to the bioreactor wherein where the cooled tank is
temperature controlled to between 30.degree. C. and 37.degree. C.
and wherein the temperature control results in maintenance of cell
viability greater than 85%.
8. The process of claim 1 wherein the broth stripper comprises an
associated broth stripper liquid reboiler, and the process further
comprises a. compressing the product rich vapor stream; b. passing
a liquid stream from the broth stripper to the broth stripper
liquid reboiler and back to the broth stripper; and c. transferring
heat, indirectly, from the compressed product rich vapor to the
liquid in the broth stripper liquid reboiler.
9. The process of claim 2 wherein the product rich vapor stream is
compressed to a pressure of between 200 mbar to 300 mbar.
10. The process of claim 2 wherein the compressing is performed by
multistage turbofans.
11. The process of claim 4 wherein between 2 and 4 turbofan stages
are provided.
12. The process of claim 2 wherein the broth stripper liquid
reboiler is positioned below theoretical distillation stages of the
vessel which are separated from an upper portion of the vessel by a
total trap-out tray.
13. The process of claim 2, wherein the transferring heat is a
process in which the product rich vapor stream is indirectly
contacted with liquid in the broth stripper liquid reboiler,
resulting in condensation of the product rich vapor stream and
evaporation of the liquid in the liquid reboiler.
14. The process of claim 1 wherein the rectification column
comprises an associated rectification column liquid reboiler, and
the process further comprises: a. compressing the rectification
column overhead stream; b. passing a liquid stream from the
rectification column to the rectification column liquid reboiler
and back to the rectification column; and c. transferring heat,
indirectly, from the compressed rectification column overhead
stream to the liquid in the rectification column liquid
reboiler.
15. The process of claim 1 further comprising passing the
rectification column overhead stream to a dehydration system.
16. A system for removing a product from a fermentation broth, the
system comprising: a. a stripper vessel comprising a feed inlet, a
vapor outlet, a liquid outlet, a bottoms recycle inlet, a reboiler
liquid outlet, and a reboiler return inlet, wherein the feed inlet
is in fluid communication with a bioreactor; b. a stripper reboiler
in fluid communication with the reboiler liquid outlet and the
reboiler return inlet; c. a first multistage mechanical
recompression unit in fluid communication with the vapor outlet of
the stripper vessel and the stripper reboiler; d. a trim condenser
in fluid communication with the stripper reboiler; e. a
rectification column in fluid communication with the trim condenser
via a rectification column inlet conduit, the rectification column
comprising an overhead outlet, a bottoms outlet, a heat exchange
outlet, and a heat exchange return inlet; f. a heat exchanger in
fluid communication with the heat exchange outlet and the heat
exchange return inlet; g. a second multistage mechanical
recompression unit in fluid communication with the overhead outlet
of the rectification column and the heat exchanger; and h. a
bottoms conduit in fluid communication with the bottoms outlet of
the rectification column and the bottoms recycle inlet of the
stripper vessel.
17. The system of claim 16 further comprising a dehydration system
in fluid communication with the heat exchanger and the second
multistage mechanical recompression unit.
18. The system of claim 16 further comprising a bottoms stream heat
exchanger in thermal communication with the bottoms conduit and the
rectification column inlet conduit.
19. The system of claim 16 further comprising a degasser in fluid
communication with the bioreactor and the feed inlet of the
stripper vessel.
20. The system of claim 16 further comprising a cooling tank in
fluid communication with the liquid outlet of the stripper vessel.
Description
CROSS REFERENCE TO A RELATED APPLICATION
[0001] The application is a continuation of U.S. application Ser.
No. 16/802,057 filed Feb. 26, 2020 which in turn is a continuation
of U.S. Pat. No. 10,610,802 issued Apr. 7, 2020 which claims the
benefit of U.S. Provisional Application No. 62/473,850 filed Mar.
20, 2017, the contents of which are hereby incorporated by
reference in their entirety.
FIELD
[0002] This invention relates to a device and associated method for
recovering at least one product from a fermentation broth. In
particular, the invention relates to the use of a vacuum
distillation vessel to recover products from a fermentation broth,
where the fermentation broth contains viable microbial biomass, and
where the recovery of product is completed in such a manner where
the viability of the microbial biomass is ensured.
BACKGROUND
[0003] Carbon dioxide (CO.sub.2) accounts for about 76% of global
greenhouse gas emissions from human activities, with methane (16%),
nitrous oxide (6%), and fluorinated gases (2%) accounting for the
balance (United States Environmental Protection Agency). The
majority of CO.sub.2 comes from the burning of fossil fuels to
produce energy, although industrial and forestry practices also
emit CO.sub.2 into the atmosphere. Reduction of greenhouse gas
emissions, particularly CO.sub.2, is critical to halt the
progression of global warming and the accompanying shifts in
climate and weather.
[0004] It has long been recognized that catalytic processes, such
as the Fischer-Tropsch process, may be used to convert gases
containing carbon dioxide (CO.sub.2), carbon monoxide (CO), and/or
hydrogen (H.sub.2), such as industrial waste gas or syngas, into a
variety of fuels and chemicals. Recently, however, gas fermentation
has emerged as an alternative platform for the biological fixation
of such gases. In particular, C1-fixing microorganisms have been
demonstrated to convert gases containing CO.sub.2, CO, and/or
H.sub.2 into products such as ethanol and 2,3-butanediol. The
production of such products may be limited, for example, by slow
microbial growth, limited gas consumption, sensitivity to toxins,
or diversion of carbon substrates into undesired by-products.
[0005] The accumulation of products can result in a reduction in
the production efficiency of the gas fermentation process. To
prevent accumulation, these products must be removed at an
effective rate. If not removed at an effective rate, these products
may have inhibitory and/or toxic effects on the C1-fixing
microorganisms. If the products accumulate to the point that the
C1-fixing microorganisms cannot survive, then the fermentation
process may have to be stopped and restarted. Prior to being
restarted, the fermenters often require cleaning. This can be a
time-consuming process.
[0006] Another pitfall commonly associated with the recovery of
products is the loss of C1-fixing microorganisms through
traditional recovery processes. To overcome the loss of viable
C1-fixing microorganisms, filtration methods have been employed.
However, over time, with traditional filtration methods,
particulate matter can build up in the filter media, which can lead
to a reduction in the filtrate flux, ultimately requiring cleaning
and/or replacement of the filter media.
[0007] In anaerobic gas fermentation, the slow growth rate of the
bacteria combined with product inhibition creates major constraints
to the productivity of the system. Vacuum distillation at
temperatures low enough for the bacteria to survive allows
selective product removal with an added cell recycle effect,
removing these constraints and allowing much higher system
productivity without the need for costly cell recycle membranes.
Accordingly, there remains a need for a system with reduced
maintenance requirements that is capable of recovering products at
an effective rate while ensuring the viability of the C1-fixing
microorganisms.
BRIEF SUMMARY
[0008] The invention provides a process for removing ethanol from a
fermentation broth, the process comprising (i) flowing a
fermentation broth comprising microbial biomass and metabolites
from a bioreactor to a vessel; (ii) partially vaporizing the
fermentation broth to produce an ethanol rich vapor stream, and an
ethanol depleted liquid stream comprising live microbial biomass;
and (iii) passing the ethanol depleted liquid stream back to the
bioreactor.
[0009] In one embodiment the fermentation broth is partially
vaporised at a temperature between 37.degree. C. and 50.degree. C.
and a pressure between 40 mbar and 100 mbar.
[0010] In one embodiment a viability of the microbial biomass in
the ethanol depleted liquid stream is greater than 85%.
[0011] In one embodiment the fermentation broth comprising
microbial biomass and metabolites is obtained from gas fermentation
of an industrial gas. Preferably the industrial gas is selected
from the group consisting of Blast furnace gas, Basic oxygen
furnace gas, gasifier syngas and PSA tail gas.
[0012] In one embodiment the fermentation broth is degassed using a
cyclonic degasser at a pressure of 0.0 bar(g) to 0.5 bar(g) prior
to the vaporization step, creating a degassed broth and an evolved
gas stream. In certain embodiments the evolved gas stream is water
scrubbed to recover product ethanol.
[0013] In one embodiment the vessel comprises a column comprising
between 8 and 12 theoretical distillation stages, wherein the
theoretical distillation stages are provided by structured packing.
In certain embodiments the fermentation broth is flowed into the
vessel at a first distillation stage. In certain embodiments, the
vessel further comprises a liquid reboiler provided below the
theoretical distillation stages, said liquid reboiler being
separated from an upper portion of the vessel by a total trap-out
tray. Preferably the vessel is separated vertically into between 2
and 4 compartments in a manner where broth from multiple fermenters
may be fed to the vessel without mixing.
[0014] In one embodiment the steam which drives separation is
provided by a process in which the vapor stream is indirectly
contacted with the reboiler liquid, resulting in condensation of
the vapor stream and evaporation of the reboiler liquid. Preferably
the vapor stream is compressed to a pressure of between 200 mbar to
300 mbar, and the compression is performed by multistage turbofans.
In preferred embodiments between 2 and 4 turbofan stages are
provided.
[0015] In one embodiment the contacting method consists of a
falling film evaporator. In embodiments wherein incomplete
condensation of the vapor stream occurs and a secondary condenser
is used to condense remaining vapor. The secondary condenser
provides cooling through indirect contact with cooling water or
chilled water.
[0016] In one embodiment the ethanol concentration in the liquid
return stream is less than 0.1% (1 g/L).
[0017] In one embodiment the liquid return stream is stored in a
cooled tank prior to return to the fermenter, the cooled tank is
temperature controlled to between 30.degree. C. and 37.degree. C.,
and the temperature control results in maintenance of cell
viability greater than 85%.
[0018] In one embodiment the concentrated ethanol stream is further
processed in a downstream Rectification column to separate ethanol
and water. In one embodiment the bottoms of the Rectification
column are recycled directly back as makeup liquid to the reboiler
area of the Vacuum stripper.
[0019] The invention provides a device, namely, a vacuum
distillation vessel, and associated method, that utilizes a vacuum
distillation vessel, for recovering at least one product from a
fermentation broth. The vacuum distillation vessel is designed for
recovering at least one product from a fermentation broth, the
fermentation broth being delivered from a bioreactor, the vacuum
distillation vessel comprising: (a) an exterior casing, defining an
inlet for receiving the fermentation broth, the fermentation broth
comprising viable microbial biomass and at least one product, an
outlet for transferring a product enriched stream, and an outlet
for transferring a product depleted stream, the product depleted
stream comprising viable microbial biomass, the product depleted
stream being transferred to the bioreactor; and (b) a separation
section located within the casing, the separation section being
bounded above by an upper tray and below by a lower tray, the
separation section defining a separation medium for providing a
plurality of theoretical distillation stages; wherein the outlet
for transferring the product enriched stream is elevated relative
to the inlet for receiving the fermentation broth, the inlet for
receiving the fermentation broth being elevated relative to the
upper tray, and the outlet for transferring the product depleted
stream being elevated relative to the lower tray.
[0020] Preferably, the vacuum distillation vessel is capable of
processing the fermentation broth at a given feed rate. The feed
rate being defined as the volume of fermentation broth per hour.
The volume of fermentation broth is the volume of fermentation
broth contained in the bioreactor. In at least one embodiment, the
vacuum distillation vessel is capable of processing the
fermentation broth at a feed rate between 0.05 and 0.5 bioreactor
volumes per hour. In certain embodiments, the feed rate is between
0.05 to 0.1, 0.05 to 0.2, 0.05 to 0.3, 0.05 to 0.4, 0.1 to 0.3, 0.1
to 0.1 to 0.5, or 0.3 to 0.5 reactor volumes per hour.
[0021] In certain instances, the fermentation broth has a given
residence time in the vacuum distillation vessel. The amount of
time the fermentation broth is within the vacuum distillation
vessel is the amount of time between the moment the fermentation
broth enters through the inlet for receiving the fermentation
broth, and when the fermentation broth exits through the outlet for
transferring the product depleted stream. Preferably, the residence
time is between 0.5 and 15 minutes. In various embodiments, the
residence time is between 0.5 and 12 minutes, 0.5 and 9 minutes,
0.5 and 6 minutes, 0.5 and 3 minutes, 2 and 15 minutes, 2 and 12
minutes, 2 and 9 minutes, or 2 and 6 minutes. In at least one
embodiment, the residence time is less than 15 minutes, less than
12 minutes, less than 9 minutes, less than 6 minutes, less than 3
minutes, less than 2 minutes, or less than 1 minute to ensure the
viability of the microorganisms.
[0022] The invention provides for the transferring of the product
depleted stream to the bioreactor through an outlet in the casing.
In at least one embodiment, the casing of the vacuum distillation
vessel is connected to the bioreactor by piping means. The product
depleted stream may be passed through the piping means from the
vacuum distillation vessel to the bioreactor. Preferably, the
bioreactor is operated under conditions for fermentation of a
C1-containing gas from an industrial process.
[0023] The vacuum distillation vessel is designed so as to
effectively remove product from the fermentation broth. The vacuum
distillation vessel utilizes a separation medium as part of the
removal process. The separation medium may be any suitable material
to provide adequate vapor-liquid contact.
[0024] In certain instances, the separation medium is provided such
that the pressure drop over the height of the vacuum distillation
vessel is less than 32 mbar. In certain instances, the pressure
drop over the height of the vacuum distillation vessel is less than
30 mbar, less than 28 mbar, less than 26 mbar, less than 24 mbar,
less than 22 mbar, less than 20 mbar, or less than 18 mbar.
[0025] In certain instances, the separation medium is defined by a
series of distillation trays. The distillation trays may be any
suitable series of distillation trays to provide adequate
vapor-liquid contact.
[0026] The separation section of the vacuum distillation vessel is
designed to provide a plurality of theoretical distillation stages
whereby an increasing amount of product is vaporized from the
fermentation broth as the fermentation broth passes through the
distillation stages. Preferably, the separation medium provides
multiple theoretical distillation stages. In certain embodiments,
the separation medium provides at least 3 theoretical distillation
stages, or at least 5 theoretical stages, or at least 6 theoretical
stages.
[0027] The vacuum distillation vessel is designed so as to ensure
the viability of the microbial biomass. By ensuring the viability
of the microbial biomass, the product depleted stream being passed
to the bioreactor may be utilized for the gas fermentation process.
Preferably, the microbial biomass viability is maintained at a
sufficiently high percentage. In certain instances, the viability
of the microbial biomass is greater than 80%, or greater than 85%,
or greater than 90%, or greater than 95%.
[0028] The vacuum distillation vessel may be designed in such a
manner that the viability of the microbial biomass is not
substantially reduced when passed through the vacuum distillation
vessel. In certain instances, the viable microbial biomass in the
product depleted stream is substantially equal to the viable
microbial biomass in the fermentation broth. Preferably, the
difference between the viability of the microbial biomass in the
product depleted stream and the viability of the microbial biomass
in the fermentation broth is less than 10%. In certain instances,
the difference is between 5 and 10%. In certain instances, the
difference is less than 5%.
[0029] The viability of the microbial biomass may be measured using
any suitable means. Preferably, the viability is measured using
flow cytometry and a live/dead assay. In certain instances, the
measurement of viability of the microbial biomass in the
fermentation broth is taken from the fermentation broth before
entering the vacuum distillation vessel. In certain instances, the
measurement of viability of the microbial biomass in the product
depleted stream is taken from the product depleted stream leaving
the vacuum distillation vessel before the product depleted stream
is passed to the bioreactor.
[0030] In certain instances, one or more variable may be changed as
a result of the viability measurement. Preferably, the one or more
variable changed as a result of the viability measurement is
selected from the group comprising: pressure, temperature,
residence time, product concentration in fermentation broth, steam
feed rate, and separation medium.
[0031] Preferably, the product depleted stream has reduced
proportions of product relative to the fermentation broth so as to
prevent, or at least mitigate, accumulation of product in the
fermentation broth. By preventing, or at least mitigating,
accumulation of product in the fermentation broth the fermentation
process may be continuous. Preferably, product is recovered from a
continuous fermentation process. In certain instances, the product
depleted stream comprises less than 1 wt. % product, or less than
0.8 wt. % product, or less than 0.6 wt. % product, or less than 0.4
wt. % product or less than 0.2 wt. % product or less than 0.1 wt. %
product.
[0032] The microorganisms in the bioreactor may be capable of
producing a variety of different products. Preferably, one or more
products recovered from the continuous fermentation process is a
low boiling fermentation product. In certain instances, product is
selected from the group consisting of ethanol, acetone,
isopropanol, butanol, ketones, methyl ethyl ketone, acetone,
2-butanol, 1-propanol, methyl acetate, ethyl acetate, butanone,
1,3-butadiene, isoprene, and isobutene. In certain instances, the
vacuum distillation vessel is designed with specific constraints
based upon the product being produced. In certain instances, the
product produced in the bioreactor is ethanol, acetone,
isopropanol, or mixtures thereof. In various instances, the product
enriched stream comprises increased proportions of ethanol,
acetone, isopropanol, or mixtures thereof, relative to the
fermentation broth. Preferably, the vacuum distillation vessel is
designed such that ethanol can be effectively removed from the
fermentation broth. In certain instances where ethanol is produced
by the microorganisms, the product enriched stream comprises
increased proportions of ethanol relative to the fermentation
broth. In certain embodiments, the vacuum distillation vessel is
designed such that acetone can be effectively removed from the
fermentation broth. In certain instances where acetone is produced
by the microorganisms, the product enriched stream comprises
increased proportions of acetone relative to the fermentation
broth. In other embodiments, the vacuum distillation vessel is
designed such that isopropanol can be effectively removed from the
fermentation broth. In certain instances where isopropanol is
produced by the microorganisms, the product enriched stream
comprises increased proportions of isopropanol relative to the
fermentation broth.
[0033] These products may be further converted to produce one or
more product. In at least one embodiment, at least one or more
product may be further converted to produce at least one component
of diesel, jet fuel, and/or gasoline. In certain instances, acetone
is further converted to produce methyl methacrylate. In certain
instances, isopropanol is further converted to produce
propylene.
[0034] To effectively remove the product from the fermentation
broth, while maintaining microorganism viability, the vacuum
distillation vessel operates at a pressure below atmospheric.
Preferably, the vacuum distillation vessel is operated at a
pressure between 40 mbar(a) and 100 mbar(a), or between 40 mbar(a)
and 80 mbar(a), or between 40 mbar(a) and 60 mbar(a), or between 50
mbar(a) and 100 mbar(a), or between 50 mbar(a) and 80 mbar(a), or
between 50 mbar(a) and 70 mbar(a), or between 60 mbar(a) and 100
mbar(a), or between 60 mbar(a) and 100 mbar(a), or between 80
mbar(a) and 100 mbar(a).
[0035] To effectively remove the product from the fermentation
broth, the vacuum distillation operates at a temperature range
capable of removing product, while ensuring the viability of the
microorganisms. In certain instances, the product is selected from
the group consisting of ethanol, acetone, and isopropanol.
Preferably, the vacuum distillation vessel is operated at a
temperature between 35.degree. C. and 50.degree. C. In one
embodiment, the temperature is between 40.degree. C. and 45.degree.
C., or between 37.degree. C. and 45.degree. C., or between
45.degree. C. and 50.degree. C. In various instances, the
temperature is less than 37.degree. C. In embodiments designed for
acetone recovery, the vacuum distillation vessel is preferably
operated at a temperature between 35.degree. C. and 50.degree. C.
In certain embodiments, for acetone recovery, the temperature is
between 35.degree. C. and 45.degree. C., or between 40.degree. C.
and 45.degree. C., or between 45.degree. C. and 50.degree. C.
[0036] In certain instances, one or more by-products are produced
by the fermentation. In certain instances, the one or more
by-products are selected from the group consisting of carboxylic
acids (i.e. acetic acid and lactic acid) and 2,3-butanediol. In
certain instances, the one or more by-products are not separated
from the fermentation broth, and are returned to the bioreactor in
the product depleted stream. Due to the continuous return of
by-products to the bioreactor, the amount of by-product in the
fermentation may accumulate. In certain instances, it is desirable
to maintain the concentration of by-products in the fermentation
broth below a predetermined level. The acceptable concentration of
by-products may be determined based on the tolerance of the microbe
to the by-product. In certain instances, it may be desirable to
provide the product depleted stream to a secondary separation means
to remove one or more by-product from the product depleted stream.
In certain embodiments the by-product is 2,3-butanediol and the
concentration of 2,3-butaendiol in the fermentation broth is
maintained below 10g/L. In certain instances, the by-product is
acetic acid and the concentration of acetic acid in the
fermentation broth is maintained below 10 g/L
[0037] In certain instances, the temperature of the product
depleted stream is elevated such that the product depleted stream
needs to be cooled prior to being passed to the bioreactor. The
temperature of the stream may have a direct effect on the viability
of the microorganism. For instance, higher temperatures may result
in a decrease in microorganism viability. To avoid the negative
effects of increased temperature, the product depleted stream may
be cooled by any suitable cooling means prior to being sent to the
bioreactor. Preferably, the temperature of the product depleted
stream is cooled to between 35.degree. C. and 40.degree. C. prior
to being returned to the bioreactor. Preferably, the fermentation
broth and the product depleted stream are kept below 45.degree. C.
to avoid the detrimental effects on viability. In one embodiment,
the temperature is between 37.degree. C. and 45.degree. C. to avoid
detrimental effects. In certain instances, the temperature is
dependent on the microorganism being used. The effect of
temperature on microorganism viability may be heightened at higher
residence times. For instance, at higher residence times, when the
temperature is above optimal, viability of the microorganisms may
decrease.
[0038] In certain instances, the fermentation broth may contain
proportions of gas. Gas in the fermentation broth has been shown to
negatively impact the performance of the vacuum distillation
vessel. This decrease in performance may be due, at least in part,
on the correlation between gas in the fermentation broth and
production of foam in the vacuum distillation vessel. To reduce the
proportions of gas in the fermentation broth, a degassing vessel
may be utilized. When utilizing a degassing vessel, the inlet for
receiving the fermentation broth may be connected by piping means
to the degassing vessel. The degassing vessel is operated under
conditions to remove at least a portion of the gas from the
fermentation broth prior to the fermentation broth being delivered
to the vacuum distillation vessel.
[0039] In certain instances, the degassing vessel is operated at
pressure. In certain instances, the degassing vessel is operated at
any pressure less than the operating pressure of the bioreactor.
Preferably, the degassing vessel is operated at a pressure between
0.0 bar(g) and 1.0 bar(g). In one embodiment, the degassing vessel
is operated at a pressure between 0.0 bar(g) and 0.5 bar(g).
Preferably, the degassing vessel removes substantially all of the
gas from the fermentation broth. In particular embodiments, the
degassing vessel removes between 0 and 100% of the gas in the
fermentation broth. In certain instances, the degassing vessel
removes more than 20%, more than 40%, more than 60%, or more than
80% of the gas from the fermentation broth. In certain instances,
the degassing vessel removes at least a portion of carbon dioxide
from the fermentation broth. In certain instances, the degassing
vessel removes at least 20%, or at least 40%, or at least 60%, or
at least 80% of carbon dioxide from the fermentation broth.
[0040] The vacuum distillation vessel may receive a vapor stream
from a reboiler. If designed to receive a vapor stream from a
reboiler, the exterior casing of the vacuum distillation vessel may
further define an inlet for receiving the vapor stream. This vapor
stream may be produced from liquid from the vacuum distillation
vessel. When utilizing liquid from the vacuum distillation vessel,
the liquid may be transferred via an outlet in the casing of the
vacuum distillation vessel. To effectively transfer the vapor
stream to the vacuum distillation vessel, the inlet for receiving
the vapor stream may be located subjacent relative to the lower
tray, and the outlet for transferring the liquid stream may be
located lower relative to the inlet for receiving the vapor
stream.
[0041] Preferably, the liquid stream is comprised substantially of
water and minimal amounts of microbial biomass. The vacuum
distillation vessel is designed to transfer viable microbial
biomass back to the bioreactor. The viable microbial biomass is
contained in the product depleted stream. The vacuum distillation
vessel transfers the product depleted to the bioreactor through the
outlet for transferring the product depleted stream. The outlet for
transferring the product depleted stream is placed above the lower
tray. Fermentation broth, containing microbial biomass, may pass
through this lower tray. This fermentation broth passing through
may then mix with the liquid in the bottom of the vacuum
distillation vessel. Preferably, only minimal amounts of microbial
biomass end up in the liquid in the bottom of the vacuum
distillation vessel. Preferably, less than 0.042 reactor volumes of
the fermentation broth, containing the microbial biomass, pass
through the lower tray per hour. In certain instances, between
0.002 and 0.042 reactor volumes of the fermentation broth,
containing the microbial biomass, pass through the lower tray per
hour. In various embodiments, less than 0.042, less than 0.037,
less than 0.032, less than 0.027, less than 0.022, less than 0.017,
less than 0.012, less than 0.007, reactor volumes of the
fermentation broth, containing the microbial biomass, pass through
the lower tray per hour. This liquid, including components of
fermentation broth containing microbial biomass, is then passed to
the reboiler to produce the vapor stream.
[0042] The vacuum distillation vessel may incorporate one or more
additional trays below the lower tray. The one or more additional
trays may provide for additional product removal. When including
one or more additional trays, the fermentation broth that passes
through the lower tray is passed to the one or more additional
trays where additional amounts of product may be recovered. After
passing through the one or more additional trays, the fermentation
broth mixes with the liquid in the bottom of the vacuum
distillation vessel. This liquid, including components of
fermentation broth containing microbial biomass, is then passed to
the reboiler to produce the vapor stream.
[0043] The vacuum distillation vessel may be separated into
multiple compartments. Preferably, when the vacuum distillation
vessel is separated into multiple compartments, the fermentation
broth within each compartment is contained such that the
fermentation broth from one compartment does not mix with
fermentation broth from another compartment. This separation may be
achieved through any suitable means. In certain instances, the
fermentation broth may be sourced from multiple bioreactors. The
product depleted stream from the fermentation broth may be sent
back to the bioreactor from which the fermentation broth was
derived. By preventing mixing between the multiple compartments,
one vacuum distillation vessel may be utilized to effectively
recover product from a plurality of bioreactors.
[0044] Preferably, the bioreactor that provides the fermentation
broth is utilized for fermentation of a C1-containing substrate.
This C1-containing substrate utilized in the fermentation process
may be sourced from one or more industrial processes. Preferably,
the industrial process is selected from the group comprising:
carbohydrate fermentation, gas fermentation, cement making, pulp
and paper making, steel making, oil refining and associated
processes, petrochemical production, coke production, anaerobic or
aerobic digestion, synthesis gas (derived from sources including
but not limited to biomass, liquid waste streams, solid waste
streams, municipal streams, fossil resources including natural gas,
coal and oil), natural gas extraction, oil extraction,
metallurgical processes, for production and/or refinement of
aluminium, copper, and/or ferroalloys, geological reservoirs and
catalytic processes (derived from the steam sources including but
not limited to steam methane reforming, steam naphtha reforming,
petroleum coke gasification, catalyst regeneration--fluid catalyst
cracking, catalyst regeneration-naphtha reforming, and dry methane
reforming).
[0045] The invention provides for a method for removing at least
one product from the fermentation broth by utilizing a vacuum
distillation vessel, the method comprising: (a) passing a
fermentation broth comprising viable microbial biomass and at least
one product from a bioreactor to a vacuum distillation vessel; (b)
partially vaporizing the fermentation broth to produce a product
enriched stream and a product depleted stream, the product depleted
stream comprising viable microbial biomass; and (c) passing the
product depleted stream back to the bioreactor. The invention may
be designed in such a manner that the viability of the microbial
biomass in the fermentation broth is ensured such that, when passed
to the bioreactor, the microbial biomass will be utilized for
fermentation of a C1-containing substrate.
[0046] Preferably, the gas in the fermentation broth is monitored
and controlled. Gas in the fermentation broth may result in a
decrease in performance of the vacuum distillation vessel. To
control the gas in the fermentation broth a degassing step may be
necessary. If the fermentation broth contains higher than
acceptable proportions of gas, fermentation broth is passed to a
degassing means prior to passing a degassed fermentation broth to
the vacuum distillation vessel.
[0047] The degassing step may be completed such that an evolved gas
stream is separated from the fermentation broth, producing a
degassed fermentation broth. The degassed fermentation broth is
then able to be partially vaporized by the vacuum distillation
vessel, producing the product enriched stream and the product
depleted stream.
[0048] The portion of gas that forms the evolved gas stream may
contain proportions of product. To prevent product loss through
removal of gas, the evolved gas stream may be sent to the
subsequent processing. In certain instances, the evolved gas stream
is passed to a water scrubber to recover at least one product. In
certain instances, the evolved gas stream may be sent to the
bioreactor.
[0049] The method may utilize a vacuum distillation vessel that
comprises a separation section located within a casing. Preferably,
the separation section located within the casing is bounded above
by an upper tray and below by a lower tray. The separation section
may provide multiple theoretical distillation stages.
[0050] The fermentation broth being processed may contain any
suitable microorganism. For example, the microorganism may be
selected from the group comprising: Escherichia coli, Saccharomyces
cerevisiae, Clostridium acetobutylicum, Clostridium beijerinckii,
Clostridium saccharbutyricum, Clostridium
saccharoperbutylacetonicum, Clostridium butyricum, Clostridium
diolis, Clostridium kluyveri, Clostridium pasterianium, Clostridium
novyi, Clostridium difficile, Clostridium thermocellum, Clostridium
cellulolyticum, Clostridium cellulovorans, Clostridium
phytofermentans, Lactococcus lactis, Bacillus subtilis, Bacillus
licheniformis, Zymomonas mobilis, Klebsiella oxytoca, Klebsiella
pneumonia, Corynebacterium glutamicum, Trichoderma reesei,
Cupriavidus necator, Pseudomonas putida, Lactobacillus plantarum,
and Methylobacterium extorquens. In certain instances, the
microorganism may be a C1-fixing bacterium selected from the group
comprising: Acetobacterium woodii, Alkalibaculum bacchii, Blautia
producta, Butyribacterium methylotrophicum, Clostridium aceticum,
Clostridium autoethanogenum, Clostridium carboxidivorans,
Clostridium coskatii, Clostridium drakei, Clostridium
formicoaceticum, Clostridium ljungdahlii, Clostridium magnum,
Clostridium ragsdalei, Clostridium scatologenes, Eubacterium
limosum, Moorella thermautotrophica, Moorella thermoacetica,
Oxobacter pfennigii, Sporomusa ovata, Sporomusa silvacetica,
Sporomusa sphaeroides, and Thermoanaerobacter kiuvi. Preferably,
the microorganism is a member of the genus Clostridium. In certain
instances, the microorganism is Clostridium autoethanogenum.
[0051] The microorganisms may be capable of producing a variety of
different products. Preferably, one or more products produced by
the microorganisms is a low boiling fermentation product. In
certain instances, product is selected from the group consisting of
ethanol, acetone, isopropanol, butanol, ketones, methyl ethyl
ketone, acetone, 2-butanol, 1-propanol, methyl acetate, ethyl
acetate, butanone, 1,3-butadiene, isoprene, and isobutene. In
certain instances, the method is optimized based upon the product
being produced. In certain instances, the product produced in the
bioreactor is ethanol. Preferably, the method is optimized such
that ethanol can be effectively removed from the fermentation
broth. In certain instances, the microorganism produces at least
one by-product. In one embodiment the at least one by-product is
selected from the group consisting of acetic acid, lactic acid and
2,3-butanediol.
BRIEF DESCRIPTION OF THE DRAWINGS
[0052] FIG. 1 is a schematic flow diagram showing the vacuum
distillation vessel, degassing vessel, and reboiler, in accordance
with one aspect of the invention.
[0053] FIG. 2 is a schematic flow diagram showing the vacuum
distillation vessel, degassing vessel, and reboiler, where the
vacuum distillation vessel includes one or more additional trays
below the lower tray, in accordance with one aspect of the
invention.
[0054] FIG. 3 is a graph showing the metabolite profile of a batch
fermentation run, in accordance with one aspect of the
invention.
[0055] FIG. 4 is a graph showing the gas uptake of the batch
fermentation run corresponding with the metabolite profile shown in
FIG. 3, in accordance with one aspect of the invention.
[0056] FIG. 5 is a graph showing the viability of the
microorganisms passing through the vacuum distillation vessel from
a bioreactor with a certain configuration, in accordance with one
aspect of the invention.
[0057] FIG. 6 is a graph showing the viability of the
microorganisms passing through the vacuum distillation vessel from
a bioreactor with a different configuration than that shown in FIG.
5, in accordance with one aspect of the invention.
[0058] FIG. 7 is a schematic flow diagram showing the system
according to one aspect of the invention.
DETAILED DESCRIPTION
[0059] The inventors have identified that by using a particularly
designed vacuum distillation vessel, at least one product, such as
ethanol, may be effectively recovered from a fermentation broth,
containing viable microbial biomass, while ensuring the viability
of the microbial biomass.
Definitions
[0060] The term "vacuum distillation vessel" is intended to
encompass a device for conducting distillation under vacuum,
wherein the liquid being distilled is enclosed at a low pressure to
reduce its boiling point. Preferably, the vacuum distillation
vessel includes a casing for enclosing a separation medium.
Preferably, the liquid being distilled is fermentation broth
comprising viable microbial biomass and at least one product. Such
fermentation broth may be sourced from a bioreactor. The bioreactor
may be used for fermentation of a C1-containing substrate.
[0061] "Casing" refers to the cover or shell protecting or
enclosing the separation medium. Preferably, the casing includes a
number of inlets and outlets for transferring liquid and/or gas.
The casing should include at least one inlet for receiving
fermentation broth, at least one outlet for transferring a product
enriched stream, and at least one outlet for transferring a product
depleted stream.
[0062] "Separation medium" is used to describe any suitable medium
capable of providing a large surface area for vapor-liquid contact,
which increases the effectiveness of the vacuum distillation
column. Such separation medium is designed to provide a plurality
of theoretical distillation stages. In at least one embodiment, the
separation medium is a series of distillation trays.
[0063] "Distillation trays" or "distillation plates" and the like
are intended to encompass plates and/or trays used to encourage
vapor-liquid contact. Tray types include sieve, valve, and bubble
cap. Sieve trays which contain holes for vapor to flow through are
used for high capacity situations providing high efficiency at a
low cost. Although less expensive, valve trays, containing holes
with opening and closing valves, have the tendency to experience
fouling due to the accumulation of material. Bubble cap trays
contain caps and are the most advanced and expensive of the three
trays, and are highly effective in some liquid flow rate
situations.
[0064] Preferably, the "upper tray" is any suitable boundary
whereby the fermentation broth may be distributed downward to the
separation medium.
[0065] Preferably, the "lower tray" is any suitable boundary to
effectuate the transfer of the product depleted stream through the
outlet in the casing.
[0066] A "theoretical distillation stage" is a hypothetical zone in
which two phases, such as the liquid and vapor phases of a
substance, establish an equilibrium with each other. The
performance of many separation processes depends on having a series
of theoretical distillation stages. The performance of a separation
device, such as a vacuum distillation vessel, may be enhanced by
providing an increased number of stages. Preferably, the separation
medium includes a sufficient number of theoretical distillation
stages to effectively remove at least one product from the
fermentation broth. Preferably, the separation medium includes
multiple theoretical distillation stages.
[0067] The term "fermentation broth" or "broth" is intended to
encompass the mixture of components including the nutrient media,
the culture of one or more microorganisms, and the one or more
products. It should be noted that the term microorganism and the
term bacteria are used interchangeably throughout the document.
[0068] "Nutrient media" or "nutrient medium" is used to describe
bacterial growth media. Generally, this term refers to a media
containing nutrients and other components appropriate for the
growth of a microbial culture. The term "nutrient" includes any
substance that may be utilised in a metabolic pathway of a
microorganism. Exemplary nutrients include potassium, B vitamins,
trace metals and amino acids.
[0069] Preferably, the fermentation broth is sent from a
"bioreactor" to the vacuum distillation vessel. The term
"bioreactor" includes a fermentation device consisting of one or
more vessels and/or towers or piping arrangements, which includes
the Continuous Stirred Tank Reactor (CSTR), Immobilized Cell
Recycles (ICR), Trickle Bed Reactor (TBR), Bubble Column, Gas Lift
Fermenter, Static Mixer, a circulated loop reactor, a membrane
reactor, such as a Hollow Fibre Membrane Bioreactor (HFM BR) or
other vessel or other device suitable for gas-liquid contact. The
reactor is preferably adapted to receive a gaseous substrate
comprising CO or CO.sub.2 or H.sub.2 or mixtures thereof. The
reactor may comprise multiple reactors (stages), either in parallel
or in series. For example, the reactor may comprise a first growth
reactor in which the bacteria are cultured and a second
fermentation reactor, to which fermentation broth from the growth
reactor may be fed and in which most of the fermentation products
may be produced.
[0070] "Gaseous substrates comprising carbon monoxide" include any
gas which contains carbon monoxide. The gaseous substrate will
typically contain a significant proportion of CO, preferably at
least about 5% to about 100% CO by volume.
[0071] While it is not necessary for the substrate to contain any
hydrogen, the presence of H.sub.2 should not be detrimental to
product formation in accordance with methods of the invention. In
particular embodiments, the presence of hydrogen results in an
improved overall efficiency of alcohol production. For example, in
particular embodiments, the substrate may comprise an approx. 2:1,
or 1:1, or 1:2 ratio of H.sub.2:CO. In one embodiment, the
substrate comprises about 30% or less H.sub.2 by volume, 20% or
less H.sub.2 by volume, about 15% or less H.sub.2 by volume or
about 10% or less H.sub.2 by volume. In other embodiments, the
substrate stream comprises low concentrations of H.sub.2, for
example, less than 5%, or less than 4%, or less than 3%, or less
than 2%, or less than 1%, or is substantially hydrogen free. The
substrate may also contain some CO.sub.2 for example, such as about
1% to about 80% CO.sub.2 by volume, or 1% to about 30% CO.sub.2 by
volume. In one embodiment, the substrate comprises less than or
equal to about 20% CO.sub.2 by volume. In particular embodiments,
the substrate comprises less than or equal to about 15% CO.sub.2 by
volume, less than or equal to about 10% CO.sub.2 by volume, less
than or equal to about 5% CO.sub.2 by volume or substantially no
CO.sub.2.
[0072] The use of a vacuum distillation vessel with a bioreactor
may increase the efficiency of the fermentation process. The terms
"increasing the efficiency", "increased efficiency" and the like,
when used in relation to a fermentation process, include, but are
not limited to, increasing one or more of the rate of growth of
microorganisms catalysing the fermentation, the growth and/or
product production rate at elevated product concentrations, the
volume of desired product produced per volume of substrate
consumed, the rate of production or level of production of the
desired product, and the relative proportion of the desired product
produced compared with other by-products of the fermentation.
[0073] Unless the context requires otherwise, the phrases
"fermenting", "fermentation process" or "fermentation reaction" and
the like, as used herein, are intended to encompass both the growth
phase and product biosynthesis phase of the microorganisms.
[0074] The fermentation process may be described as either "batch"
or "continuous". "Batch fermentation" is used to describe a
fermentation process where the bioreactor is filled with raw
material, i.e. the carbon source, along with microorganisms, where
the products remain in the bioreactor until fermentation is
completed. In a "batch" process, after fermentation is completed,
the products are extracted and the bioreactor is cleaned before the
next "batch" is started. "Continuous fermentation" is used to
describe a fermentation process where the fermentation process is
extended for longer periods of time, and product and/or metabolite
is extracted during fermentation. Preferably, the vacuum
distillation vessel removes product from a "continuous
fermentation" process.
[0075] A "microorganism" is a microscopic organism, especially a
bacterium, archea, virus, or fungus. The microorganism of the
invention is typically a bacterium. As used herein, recitation of
"microorganism" should be taken to encompass "bacterium."
[0076] "Viability" or "viability of the microbial biomass" and the
like refers to the ratio of microorganisms that are alive, capable
of living, developing, or reproducing to those that are not. For
example, viable microbial biomass in a vacuum distillation vessel
may refer to the ratio of live/dead microorganisms within the
vacuum distillation vessel. The invention may be designed so that
the viability of the microbial biomass is maintained at a minimum
viability. In at least one embodiment, the viability of the
microbial biomass is at least about 85%. In at least one
embodiment, the viable microbial biomass is returned from the
vacuum distillation vessel back to the bioreactor.
[0077] "Effective rate of product recovery" and the like refers to
the rate at which product can be recovered from the fermentation
broth so as to prevent, or at least mitigate, the toxic and/or
inhibitory effects associated with product accumulation. The
invention may be designed so that the effective rate of product
recovery is such that the viability of the microbial biomass is
maintained above a desired threshold. The invention may be designed
so that the level of product concentration in the broth is kept
below a desired threshold. For example, the invention may be
designed such that the ethanol concentration in the fermentation
broth is kept below 40 g/L. In certain instances, the ethanol
concentration in the fermentation broth is kept between 25 to 35
g/L. In particular instances, the ethanol concentration in the
fermentation broth is less than 30 g/L, less than 35 g/L, or less
than 38 g/L. Preferably, the ethanol concentration in the
fermentation broth is less than the concentration that may result
in inhibition of the microorganism. In particular instances, the
inhibition may be dependent on the microorganism being used and the
product being produced.
[0078] The vacuum distillation vessel may pass the product depleted
stream to a "cooling means" prior to the product depleted stream
being passed to the bioreactor. The term "cooling means" may
describe any suitable device or process capable of reducing the
temperature of the product depleted stream.
[0079] The microorganisms in bioreactor may be modified from a
naturally-occurring microorganism. A "parental microorganism" is a
microorganism used to generate a microorganism of the invention.
The parental microorganism may be a naturally-occurring
microorganism (i.e., a wild-type microorganism) or a microorganism
that has been previously modified (i.e., a mutant or recombinant
microorganism). The microorganism of the invention may be modified
to express or overexpress one or more enzymes that were not
expressed or overexpressed in the parental microorganism.
Similarly, the microorganism of the invention may be modified to
contain one or more genes that were not contained by the parental
microorganism. The microorganism of the invention may also be
modified to not express or to express lower amounts of one or more
enzymes that were expressed in the parental microorganism. In one
embodiment, the parental microorganism is Clostridium
autoethanogenum, Clostridium ljungdahlii, or Clostridium ragsdalei.
In a preferred embodiment, the parental microorganism is
Clostridium autoethanogenum LZ1561, which was deposited on Jun. 7,
2010 with Deutsche Sammlung von Mikroorganismen and Zellkulturen
GmbH (DSMZ) located at Inhoffenstra 7B, D-38124 Braunschwieg,
Germany on Jun. 7, 2010 under the terms of the Budapest Treaty and
accorded accession number DSM23693. This strain is described in
International Patent Application No. PCT/NZ2011/000144, which
published as WO 2012/015317.
[0080] "Wood-Ljungdahl" refers to the Wood-Ljungdahl pathway of
carbon fixation as described, i.e., by Ragsdale, Biochim Biophys
Acta, 1784: 1873-1898, 2008. "Wood-Ljungdahl microorganisms"
refers, predictably, to microorganisms containing the
Wood-Ljungdahl pathway. Generally, the microorganism of the
invention contains a native Wood-Ljungdahl pathway. Herein, a
Wood-Ljungdahl pathway may be a native, unmodified Wood-Ljungdahl
pathway or it may be a Wood-Ljungdahl pathway with some degree of
genetic modification (i.e., overexpression, heterologous
expression, knockout, etc.) so long as it still functions to
convert CO, CO.sub.2, and/or H.sub.2 to acetyl-CoA.
[0081] "C1" refers to a one-carbon molecule, for example, CO,
CO.sub.2, CH.sub.4, or CH.sub.3OH. "C1-oxygenate" refers to a
one-carbon molecule that also comprises at least one oxygen atom,
for example, CO, CO.sub.2, or CH.sub.3OH. "C1-carbon source" refers
a one carbon-molecule that serves as a partial or sole carbon
source for the microorganism of the invention. For example, a
C1-carbon source may comprise one or more of CO, CO.sub.2,
CH.sub.4, CH.sub.3OH, or CH.sub.2O.sub.2. Preferably, the C1-carbon
source comprises one or both of CO and CO.sub.2. A "C1-fixing
microorganism" is a microorganism that has the ability to produce
one or more products from a C1-carbon source. Typically, the
microorganism of the invention is a C1-fixing bacterium.
[0082] An "anaerobe" is a microorganism that does not require
oxygen for growth. An anaerobe may react negatively or even die if
oxygen is present above a certain threshold. However, some
anaerobes are capable of tolerating low levels of oxygen (i.e.,
0.000001-5% oxygen). Typically, the microorganism of the invention
is an anaerobe.
[0083] "Acetogens" are obligately anaerobic bacteria that use the
Wood-Ljungdahl pathway as their main mechanism for energy
conservation and for synthesis of acetyl-CoA and acetyl-CoA-derived
products, such as acetate (Ragsdale, Biochim Biophys Acta, 1784:
1873-1898, 2008). In particular, acetogens use the Wood-Ljungdahl
pathway as a (1) mechanism for the reductive synthesis of
acetyl-CoA from CO.sub.2, (2) terminal electron-accepting, energy
conserving process, (3) mechanism for the fixation (assimilation)
of CO.sub.2 in the synthesis of cell carbon (Drake, Acetogenic
Prokaryotes, In: The Prokaryotes, 3.sup.rd edition, p. 354, New
York, N.Y., 2006). All naturally occurring acetogens are C1-fixing,
anaerobic, autotrophic, and non-methanotrophic. Typically, the
microorganism of the invention is an acetogen.
[0084] An "ethanologen" is a microorganism that produces or is
capable of producing ethanol. Typically, the microorganism of the
invention is an ethanologen.
[0085] An "autotroph" is a microorganism capable of growing in the
absence of organic carbon. Instead, autotrophs use inorganic carbon
sources, such as CO and/or CO.sub.2. Typically, the microorganism
of the invention is an autotroph.
[0086] A "carboxydotroph" is a microorganism capable of utilizing
CO as a sole source of carbon and energy. Typically, the
microorganism of the invention is a carboxydotroph.
[0087] A "methanotroph" is a microorganism capable of utilizing
methane as a sole source of carbon and energy. In certain
embodiments, the microorganism of the invention is a methanotroph
or is derived from a methanotroph. In other embodiments, the
microorganism of the invention is not a methanotroph or is not
derived from a methanotroph.
[0088] "Substrate" refers to a carbon and/or energy source for the
microorganism of the invention. Typically, the substrate is gaseous
and comprises a C1-carbon source, for example, CO, CO.sub.2, and/or
CH.sub.4. Preferably, the substrate comprises a C1-carbon source of
CO or CO+CO.sub.2. The substrate may further comprise other
non-carbon components, such as H.sub.2, N.sub.2, or electrons.
[0089] The term "co-substrate" refers to a substance that, while
not necessarily being the primary energy and material source for
product synthesis, can be utilised for product synthesis when added
to another substrate, such as the primary substrate.
[0090] Although the substrate is typically gaseous, the substrate
may also be provided in alternative forms. For example, the
substrate may be dissolved in a liquid saturated with a
CO-containing gas using a microbubble dispersion generator. By way
of further example, the substrate may be adsorbed onto a solid
support.
[0091] The substrate and/or C1-carbon source may be a waste gas
obtained as a by-product of an industrial process or from some
other source, such as from automobile exhaust fumes or biomass
gasification. In certain embodiments, the industrial process is
selected from the group consisting gas emissions from carbohydrate
fermentation, gas fermentation, gas emissions from cement making,
pulp and paper making, steel making, oil refining and associated
processes, petrochemical production, coke production, anaerobic or
aerobic digestion, synthesis gas (derived from sources including
but not limited to biomass, liquid waste streams, solid waste
streams, municipal streams, fossil resources including natural gas,
coal and oil), natural gas extraction, oil extraction,
metallurgical processes, for production and/or refinement of
aluminium, copper, and/or ferroalloys, geological reservoirs, and
catalytic processes (derived from the steam sources including but
not limited to steam methane reforming, steam naphtha reforming,
petroleum coke gasification, catalyst regeneration--fluid catalyst
cracking, catalyst regeneration-naphtha reforming, and dry methane
reforming). In these embodiments, the substrate and/or C1-carbon
source may be captured from the industrial process before it is
emitted into the atmosphere, using any convenient method.
[0092] The microorganism of the invention may be cultured with the
gas stream to produce one or more products. For instance, the
microorganism of the invention may produce or may be engineered to
produce ethanol (WO 2007/117157), acetate (WO 2007/117157), butanol
(WO 2008/115080 and WO 2012/053905), butyrate (WO 2008/115080),
2,3-butanediol (WO 2009/151342 and WO 2016/094334), lactate (WO
2011/112103), butene (WO 2012/024522), butadiene (WO 2012/024522),
methyl ethyl ketone (2-butanone) (WO 2012/024522 and WO
2013/185123), ethylene (WO 2012/026833), acetone (WO 2012/115527),
isopropanol (WO 2012/115527), lipids (WO 2013/036147),
3-hydroxypropionate (3-HP) (WO 2013/180581), terpenes, including
isoprene (WO 2013/180584), fatty acids (WO 2013/191567), 2-butanol
(WO 2013/185123), 1,2-propanediol (WO 2014/036152), 1-propanol (WO
2014/0369152), chorismate-derived products (WO 2016/191625),
3-hydroxybutyrate (WO 2017/066498), and 1,3-butanediol (WO
2017/0066498). In certain embodiments, microbial biomass itself may
be considered a product.
[0093] A "native product" is a product produced by a genetically
unmodified microorganism. For example, ethanol, acetate, and
2,3-butanediol are native products of Clostridium autoethanogenum,
Clostridium ljungdahlii, and Clostridium ragsdalei. A "non-native
product" is a product that is produced by a genetically modified
microorganism, but is not produced by a genetically unmodified
microorganism from which the genetically modified microorganism is
derived.
[0094] "Selectivity" refers to the ratio of the production of a
target product to the production of all fermentation products
produced by a microorganism. The microorganism of the invention may
be engineered to produce products at a certain selectivity or at a
minimum selectivity. In one embodiment, a target product accounts
for at least about 5%, 10%, 15%, 20%, 30%, 50%, 75%, or 95% of all
fermentation products produced by the microorganism of the
invention. In one embodiment, the target product accounts for at
least 10% of all fermentation products produced by the
microorganism of the invention, such that the microorganism of the
invention has a selectivity for the target product of at least 10%.
In another embodiment, the target product accounts for at least 30%
of all fermentation products produced by the microorganism of the
invention, such that the microorganism of the invention has a
selectivity for the target product of at least 30%.
[0095] The vacuum distillation vessel is capable of recovering one
or more "low boiling fermentation product." A "low boiling
fermentation product" is a product that is volatile. These products
may include, but are not limited to, ethanol, acetone, isopropanol,
butanol, ketones, methyl ethyl ketone, 2-butanol, 1-propanol,
methyl acetate, ethyl acetate, butanone, 1,3-butadiene, isoprene,
and isobutene.
[0096] The culture is generally maintained in an aqueous culture
medium that contains nutrients, vitamins, and/or minerals
sufficient to permit growth of the microorganism. Preferably the
aqueous culture medium is an anaerobic microbial growth medium,
such as a minimal anaerobic microbial growth medium. Suitable media
are well known in the art.
[0097] The culture/fermentation should desirably be carried out
under appropriate conditions for production of the target product.
Typically, the culture/fermentation is performed under anaerobic
conditions. Reaction conditions to consider include pressure (or
partial pressure), temperature, gas flow rate, liquid flow rate,
media pH, media redox potential, agitation rate (if using a
continuous stirred tank reactor), inoculum level, maximum gas
substrate concentrations to ensure that gas in the liquid phase
does not become limiting, and maximum product concentrations to
avoid product inhibition. In particular, the rate of introduction
of the substrate may be controlled to ensure that the concentration
of gas in the liquid phase does not become limiting, since products
may be consumed by the culture under gas-limited conditions.
[0098] Operating a bioreactor at elevated pressures allows for an
increased rate of gas mass transfer from the gas phase to the
liquid phase. Accordingly, it is generally preferable to perform
the culture/fermentation at pressures higher than atmospheric
pressure. Also, since a given gas conversion rate is, in part, a
function of the substrate retention time and retention time
dictates the required volume of a bioreactor, the use of
pressurized systems can greatly reduce the volume of the bioreactor
required and, consequently, the capital cost of the
culture/fermentation equipment. This, in turn, means that the
retention time, defined as the liquid volume in the bioreactor
divided by the input gas flow rate, can be reduced when bioreactors
are maintained at elevated pressure rather than atmospheric
pressure. The optimum reaction conditions will depend partly on the
particular microorganism used. However, in general, it is
preferable to operate the fermentation at a pressure higher than
atmospheric pressure. Also, since a given gas conversion rate is in
part a function of substrate retention time and achieving a desired
retention time in turn dictates the required volume of a
bioreactor, the use of pressurized systems can greatly reduce the
volume of the bioreactor required, and consequently, the capital
cost of the fermentation equipment.
[0099] The term "non-naturally occurring" when used in reference to
a microorganism is intended to mean that the microorganism has at
least one genetic modification not normally found in a naturally
occurring strain of the referenced species, including wild-type
strains of the referenced species.
[0100] The terms "genetic modification," "genetic alteration," or
"genetic engineering" broadly refer to manipulation of the genome
or nucleic acids of a microorganism by the hand of man. Likewise,
the terms "genetically modified," "genetically altered," or
"genetically engineered" refers to a microorganism containing such
a genetic modification, genetic alteration, or genetic engineering.
These terms may be used to differentiate a lab-generated
microorganism from a naturally-occurring microorganism. Methods of
genetic modification of include, for example, heterologous gene
expression, gene or promoter insertion or deletion, nucleic acid
mutation, altered gene expression or inactivation, enzyme
engineering, directed evolution, knowledge-based design, random
mutagenesis methods, gene shuffling, and codon optimization.
[0101] "Recombinant" indicates that a nucleic acid, protein, or
microorganism is the product of genetic modification, engineering,
or recombination. Generally, the term "recombinant" refers to a
nucleic acid, protein, or microorganism that contains or is encoded
by genetic material derived from multiple sources, such as two or
more different strains or species of microorganisms. As used
herein, the term "recombinant" may also be used to describe a
microorganism that comprises a mutated nucleic acid or protein,
including a mutated form of an endogenous nucleic acid or
protein.
[0102] "Wild type" refers to the typical form of an organism,
strain, gene, or characteristic as it occurs in nature, as
distinguished from mutant or variant forms.
[0103] "Endogenous" refers to a nucleic acid or protein that is
present or expressed in the wild-type or parental microorganism
from which the microorganism of the invention is derived. For
example, an endogenous gene is a gene that is natively present in
the wild-type or parental microorganism from which the
microorganism of the invention is derived. In one embodiment, the
expression of an endogenous gene may be controlled by an exogenous
regulatory element, such as an exogenous promoter.
[0104] "Exogenous" refers to a nucleic acid or protein that is not
present in the wild-type or parental microorganism from which the
microorganism of the invention is derived. In one embodiment, an
exogenous gene or enzyme may be derived from a heterologous (i.e.,
different) strain or species and introduced to or expressed in the
microorganism of the invention. In another embodiment, an exogenous
gene or enzyme may be artificially or recombinantly created and
introduced to or expressed in the microorganism of the invention.
Exogenous nucleic acids may be adapted to integrate into the genome
of the microorganism of the invention or to remain in an
extra-chromosomal state in the microorganism of the invention, for
example, in a plasmid.
[0105] The terms "polynucleotide," "nucleotide," "nucleotide
sequence," "nucleic acid," and "oligonucleotide" are used
interchangeably. They refer to a polymeric form of nucleotides of
any length, either deoxyribonucleotides or ribonucleotides, or
analogs thereof. Polynucleotides may have any three dimensional
structure, and may perform any function, known or unknown. The
following are non-limiting examples of polynucleotides: coding or
non-coding regions of a gene or gene fragment, loci (locus) defined
from linkage analysis, exons, introns, messenger RNA (mRNA),
transfer RNA, ribosomal RNA, short interfering RNA (siRNA),
short-hairpin RNA (shRNA), micro-RNA (miRNA), ribozymes, cDNA,
recombinant polynucleotides, branched polynucleotides, plasmids,
vectors, isolated DNA of any sequence, isolated RNA of any
sequence, nucleic acid probes, and primers. A polynucleotide may
comprise one or more modified nucleotides, such as methylated
nucleotides or nucleotide analogs. If present, modifications to the
nucleotide structure may be imparted before or after assembly of
the polymer. The sequence of nucleotides may be interrupted by
non-nucleotide components. A polynucleotide may be further modified
after polymerization, such as by conjugation with a labeling
component.
[0106] As used herein, "expression" refers to the process by which
a polynucleotide is transcribed from a DNA template (such as into
and mRNA or other RNA transcript) and/or the process by which a
transcribed mRNA is subsequently translated into peptides,
polypeptides, or proteins. Transcripts and encoded polypeptides may
be collectively referred to as "gene products."
[0107] The terms "polypeptide", "peptide," and "protein" are used
interchangeably herein to refer to polymers of amino acids of any
length. The polymer may be linear or branched, it may comprise
modified amino acids, and it may be interrupted by non-amino acids.
The terms also encompass an amino acid polymer that has been
modified; for example, disulfide bond formation, glycosylation,
lipidation, acetylation, phosphorylation, or any other
manipulation, such as conjugation with a labeling component. As
used herein, the term "amino acid" includes natural and/or
unnatural or synthetic amino acids, including glycine and both the
D or L optical isomers, and amino acid analogs and
peptidomimetics.
[0108] "Enzyme activity," or simply "activity," refers broadly to
enzymatic activity, including, but not limited, to the activity of
an enzyme, the amount of an enzyme, or the availability of an
enzyme to catalyze a reaction. Accordingly, "increasing" enzyme
activity includes increasing the activity of an enzyme, increasing
the amount of an enzyme, or increasing the availability of an
enzyme to catalyze a reaction. Similarly, "decreasing" enzyme
activity includes decreasing the activity of an enzyme, decreasing
the amount of an enzyme, or decreasing the availability of an
enzyme to catalyze a reaction.
[0109] "Mutated" refers to a nucleic acid or protein that has been
modified in the microorganism of the invention compared to the
wild-type or parental microorganism from which the microorganism of
the invention is derived. In one embodiment, the mutation may be a
deletion, insertion, or substitution in a gene encoding an enzyme.
In another embodiment, the mutation may be a deletion, insertion,
or substitution of one or more amino acids in an enzyme.
[0110] In particular, a "disruptive mutation" is a mutation that
reduces or eliminates (i.e., "disrupts") the expression or activity
of a gene or enzyme. The disruptive mutation may partially
inactivate, fully inactivate, or delete the gene or enzyme. The
disruptive mutation may be a knockout (KO) mutation. The disruptive
mutation may be any mutation that reduces, prevents, or blocks the
biosynthesis of a product produced by an enzyme. The disruptive
mutation may include, for example, a mutation in a gene encoding an
enzyme, a mutation in a genetic regulatory element involved in the
expression of a gene encoding an enzyme, the introduction of a
nucleic acid which produces a protein that reduces or inhibits the
activity of an enzyme, or the introduction of a nucleic acid (e.g.,
antisense RNA, siRNA, CRISPR) or protein which inhibits the
expression of an enzyme. The disruptive mutation may be introduced
using any method known in the art.
[0111] Introduction of a disruptive mutation results in a
microorganism of the invention that produces no target product or
substantially no target product or a reduced amount of target
product compared to the parental microorganism from which the
microorganism of the invention is derived. For example, the
microorganism of the invention may produce no target product or at
least about 1%, 3%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,
90%, or 95% less target product than the parental microorganism.
For example, the microorganism of the invention may produce less
than about 0.001, 0.01, 0.10, 0.30, 0.50, or 1.0 g/L target
product.
[0112] "Codon optimization" refers to the mutation of a nucleic
acid, such as a gene, for optimized or improved translation of the
nucleic acid in a particular strain or species. Codon optimization
may result in faster translation rates or higher translation
accuracy. In a preferred embodiment, the genes of the invention are
codon optimized for expression in Clostridium, particularly
Clostridium autoethanogenum, Clostridium ljungdahlii, or
Clostridium ragsdalei. In a further preferred embodiment, the genes
of the invention are codon optimized for expression in Clostridium
autoethanogenum LZ1561, which is deposited under DSMZ accession
number DSM23693.
[0113] "Overexpressed" refers to an increase in expression of a
nucleic acid or protein in the microorganism of the invention
compared to the wild-type or parental microorganism from which the
microorganism of the invention is derived. Overexpression may be
achieved by any means known in the art, including modifying gene
copy number, gene transcription rate, gene translation rate, or
enzyme degradation rate.
[0114] The term "variants" includes nucleic acids and proteins
whose sequence varies from the sequence of a reference nucleic acid
and protein, such as a sequence of a reference nucleic acid and
protein disclosed in the prior art or exemplified herein. The
invention may be practiced using variant nucleic acids or proteins
that perform substantially the same function as the reference
nucleic acid or protein. For example, a variant protein may perform
substantially the same function or catalyze substantially the same
reaction as a reference protein. A variant gene may encode the same
or substantially the same protein as a reference gene. A variant
promoter may have substantially the same ability to promote the
expression of one or more genes as a reference promoter.
[0115] Such nucleic acids or proteins may be referred to herein as
"functionally equivalent variants." By way of example, functionally
equivalent variants of a nucleic acid may include allelic variants,
fragments of a gene, mutated genes, polymorphisms, and the like.
Homologous genes from other microorganisms are also examples of
functionally equivalent variants. These include homologous genes in
species such as Clostridium acetobutylicum, Clostridium
beijerinckii, or Clostridium ljungdahlii, the details of which are
publicly available on websites such as Genbank or NCBI.
Functionally equivalent variants also include nucleic acids whose
sequence varies as a result of codon optimization for a particular
microorganism. A functionally equivalent variant of a nucleic acid
will preferably have at least approximately 70%, approximately 80%,
approximately 85%, approximately 90%, approximately 95%,
approximately 98%, or greater nucleic acid sequence identity
(percent homology) with the referenced nucleic acid. A functionally
equivalent variant of a protein will preferably have at least
approximately 70%, approximately 80%, approximately 85%,
approximately 90%, approximately 95%, approximately 98%, or greater
amino acid identity (percent homology) with the referenced protein.
The functional equivalence of a variant nucleic acid or protein may
be evaluated using any method known in the art.
[0116] "Complementarity" refers to the ability of a nucleic acid to
form hydrogen bond(s) with another nucleic acid sequence by either
traditional Watson-Crick or other non-traditional types. A percent
complementarity indicates the percentage of residues in a nucleic
acid molecule which can form hydrogen bonds (e.g., Watson-Crick
base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7,
8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100%
complementary). "Perfectly complementary" means that all the
contiguous residues of a nucleic acid sequence will hydrogen bond
with the same number of contiguous residues in a second nucleic
acid sequence. "Substantially complementary" as used herein refers
to a degree of complementarity that is at least 60%, 65%, 70%, 75%,
80%, 85%, 90%, 95%. 97%, 98%, 99%, or 100% over a region of 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30,
35, 40, 45, 50, or more nucleotides, or refers to two nucleic acids
that hybridize under stringent conditions.
[0117] As used herein, "stringent conditions" for hybridization
refer to conditions under which a nucleic acid having
complementarity to a target sequence predominantly hybridizes with
the target sequence, and substantially does not hybridize to
non-target sequences. Stringent conditions are generally
sequence-dependent, and vary depending on a number of factors. In
general, the longer the sequence, the higher the temperature at
which the sequence specifically hybridizes to its target sequence.
Non-limiting examples of stringent conditions are well known in the
art (e.g., Tijssen, Laboratory techniques in biochemistry and
molecular biology-hybridization with nucleic acid probes, Second
Chapter "Overview of principles of hybridization and the strategy
of nucleic acid probe assay," Elsevier, N.Y., 1993).
[0118] "Hybridization" refers to a reaction in which one or more
polynucleotides react to form a complex that is stabilized via
hydrogen bonding between the bases of the nucleotide residues. The
hydrogen bonding may occur by Watson Crick base pairing, Hoogstein
binding, or in any other sequence specific manner. The complex may
comprise two strands forming a duplex structure, three or more
strands forming a multi stranded complex, a single self-hybridizing
strand, or any combination of these. A hybridization reaction may
constitute a step in a more extensive process, such as the
initiation of PCR, or the cleavage of a polynucleotide by an
enzyme. A sequence capable of hybridizing with a given sequence is
referred to as the "complement" of the given sequence.
[0119] Nucleic acids may be delivered to a microorganism of the
invention using any method known in the art. For example, nucleic
acids may be delivered as naked nucleic acids or may be formulated
with one or more agents, such as liposomes. The nucleic acids may
be DNA, RNA, cDNA, or combinations thereof, as is appropriate.
Restriction inhibitors may be used in certain embodiments.
Additional vectors may include plasmids, viruses, bacteriophages,
cosmids, and artificial chromosomes. In a preferred embodiment,
nucleic acids are delivered to the microorganism of the invention
using a plasmid. By way of example, transformation (including
transduction or transfection) may be achieved by electroporation,
ultrasonication, polyethylene glycol-mediated transformation,
chemical or natural competence, protoplast transformation, prophage
induction, or conjugation. In certain embodiments having active
restriction enzyme systems, it may be necessary to methylate a
nucleic acid before introduction of the nucleic acid into a
microorganism.
[0120] Furthermore, nucleic acids may be designed to comprise a
regulatory element, such as a promoter, to increase or otherwise
control expression of a particular nucleic acid. The promoter may
be a constitutive promoter or an inducible promoter. Ideally, the
promoter is a Wood-Ljungdahl pathway promoter, a ferredoxin
promoter, a pyruvate:ferredoxin oxidoreductase promoter, an Rnf
complex operon promoter, an ATP synthase operon promoter, or a
phosphotransacetylase/acetate kinase operon promoter.
[0121] A "microorganism" is a microscopic organism, especially a
bacterium, archea, virus, or fungus. The microorganism of the
invention is typically a bacterium. As used herein, recitation of
"microorganism" should be taken to encompass "bacterium."
[0122] A "parental microorganism" is a microorganism used to
generate a microorganism of the invention. The parental
microorganism may be a naturally-occurring microorganism (i.e., a
wild-type microorganism) or a microorganism that has been
previously modified (i.e., a mutant or recombinant microorganism).
The microorganism of the invention may be modified to express or
overexpress one or more enzymes that were not expressed or
overexpressed in the parental microorganism. Similarly, the
microorganism of the invention may be modified to contain one or
more genes that were not contained by the parental microorganism.
The microorganism of the invention may also be modified to not
express or to express lower amounts of one or more enzymes that
were expressed in the parental microorganism. In one embodiment,
the parental microorganism is Clostridium autoethanogenum,
Clostridium ljungdahlii, or Clostridium ragsdalei. In a preferred
embodiment, the parental microorganism is Clostridium
autoethanogenum LZ1561, which was deposited on Jun. 7, 2010 with
Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH (DSMZ)
located at Inhoffenstra 7B, D-38124 Braunschwieg, Germany on June
7, 2010 under the terms of the Budapest Treaty and accorded
accession number DSM23693.
[0123] The term "derived from" indicates that a nucleic acid,
protein, or microorganism is modified or adapted from a different
(e.g., a parental or wild-type) nucleic acid, protein, or
microorganism, so as to produce a new nucleic acid, protein, or
microorganism. Such modifications or adaptations typically include
insertion, deletion, mutation, or substitution of nucleic acids or
genes. Generally, the microorganism of the invention is derived
from a parental microorganism. In one embodiment, the microorganism
of the invention is derived from Clostridium autoethanogenum,
Clostridium ljungdahlii, or Clostridium ragsdalei. In a preferred
embodiment, the microorganism of the invention is derived from
Clostridium autoethanogenum LZ1561, which is deposited under DSMZ
accession number DSM23693.
[0124] The microorganism of the invention may be further classified
based on functional characteristics. For example, the microorganism
of the invention may be or may be derived from a C1-fixing
microorganism, an anaerobe, an acetogen, an ethanologen, a
carboxydotroph, and/or a methanotroph. Table 1 provides a
representative list of microorganisms and identifies their
functional characteristics.
TABLE-US-00001 TABLE 1 C1-fixing Anaerobe Acetogen Ethanologen
Autotroph Carboxydotroph Methanotroph Acetobacterium woodii + + +
+/- .sup.1 - - - Alkalibaculum bacchii + + + + + + - Blautia
producta + + + - + + - Butyribacterium methylotrophicum + + + + + +
- Clostridium aceticum + + + - + + - Clostridium autoethanogenum +
+ + + + + - Clostridium carboxidivorans + + + + + + - Clostridium
coskatii + + + + + + - Clostridium drakei + + + - + + - Clostridium
formicoaceticum + + + - + + - Clostridium ljungdahlii + + + + + + -
Clostridium magnum + + + - + +/- .sup.2 - Clostridium ragsdalei + +
+ + + + - Clostridium scatologenes + + + - + + - Eubacterium
limosum + + + - + + - Moorella thermautotrophica + + + + + + -
Moorella thermoacetica (formerly + + + .sup. - 3 + + - Clostridium
thermoaceticum) Oxobacter pfennigii + + + - + + - Sporomusa ovata +
+ + - + +/- .sup.4 - Sporomusa silvacetica + + + - + +/- .sup.5 -
Sporomusa sphaeroides + + + - + +/- .sup.6 - Thermoanaerobacter
kiuvi + + + - + - - .sup.1 Acetobacterium woodi can produce ethanol
from fructose, but not from gas. .sup.2 It has not been
investigated whether Clostridium magnum can grow on CO. .sup.3 One
strain of Moorella thermoacetica, Moorella sp. HUC22-1, has been
reported to produce ethanol from gas. .sup.4 It has not been
investigated whether Sporomusa ovata can grow on CO. .sup.5 It has
not been investigated whether Sporomusa silvacetica can grow on CO.
.sup.6 It has not been investigated whether Sporomusa sphaeroides
can grow on CO.
[0125] In a preferred embodiment, the microorganism of the
invention is derived from a C1-fixing microorganism identified in
Table 1. In a preferred embodiment, the microorganism of the
invention is derived from an acetogen identified in Table 1. In a
preferred embodiment, the microorganism of the invention is derived
from an ethanologen identified in Table 1. In a preferred
embodiment, the microorganism of the invention is derived from an
autotroph identified in Table 1. In a preferred embodiment, the
microorganism of the invention is derived from a carboxydotroph
identified in Table 1. More broadly, the microorganism of the
invention may be derived from any genus or species identified in
Table 1.
[0126] In a preferred embodiment, the microorganism of the
invention is derived from the cluster of Clostridia comprising the
species Clostridium autoethanogenum, Clostridium ljungdahlii, and
Clostridium ragsdalei. These species were first reported and
characterized by Abrini, Arch Microbiol, 161: 345-351, 1994
(Clostridium autoethanogenum), Tanner, Int J System Bacteriol, 43:
232-236, 1993 (Clostridium ljungdahlii), and Huhnke, WO 2008/028055
(Clostridium ragsdalei).
[0127] These three species have many similarities. In particular,
these species are all C1-fixing, anaerobic, acetogenic,
ethanologenic, and carboxydotrophic members of the genus
Clostridium. These species have similar genotypes and phenotypes
and modes of energy conservation and fermentative metabolism.
Moreover, these species are clustered in clostridial rRNA homology
group I with 16S rRNA DNA that is more than 99% identical, have a
DNA G+C content of about 22-30 mol%, are gram-positive, have
similar morphology and size (logarithmic growing cells between
0.5-0.7.times.3-5 .mu.m), are mesophilic (grow optimally at
30-37.degree. C.), have similar pH ranges of about 4-7.5 (with an
optimal pH of about 5.5-6), lack cytochromes, and conserve energy
via an Rnf complex. Also, reduction of carboxylic acids into their
corresponding alcohols has been shown in these species (Perez,
Biotechnol Bioeng, 110:1066-1077, 2012). Importantly, these species
also all show strong autotrophic growth on CO-containing gases,
produce ethanol and acetate (or acetic acid) as main fermentation
products, and produce small amounts of 2,3-butanediol and lactic
acid under certain conditions.
[0128] However, these three species also have a number of
differences. These species were isolated from different sources:
Clostridium autoethanogenum from rabbit gut, Clostridium
ljungdahlii from chicken yard waste, and Clostridium ragsdalei from
freshwater sediment. These species differ in utilization of various
sugars (e.g., rhamnose, arabinose), acids (e.g., gluconate,
citrate), amino acids (e.g., arginine, histidine), and other
substrates (e.g., betaine, butanol). Moreover, these species differ
in auxotrophy to certain vitamins (e.g., thiamine, biotin). These
species have differences in nucleic and amino acid sequences of
Wood-Ljungdahl pathway genes and proteins, although the general
organization and number of these genes and proteins has been found
to be the same in all species (Kopke, Curr Opin Biotechnol, 22:
320-325, 2011).
[0129] Thus, in summary, many of the characteristics of Clostridium
autoethanogenum, Clostridium ljungdahlii, or Clostridium ragsdalei
are not specific to that species, but are rather general
characteristics for this cluster of C1-fixing, anaerobic,
acetogenic, ethanologenic, and carboxydotrophic members of the
genus Clostridium. However, since these species are, in fact,
distinct, the genetic modification or manipulation of one of these
species may not have an identical effect in another of these
species. For instance, differences in growth, performance, or
product production may be observed.
[0130] The microorganism of the invention may also be derived from
an isolate or mutant of Clostridium autoethanogenum, Clostridium
ljungdahlii, or Clostridium ragsdalei. Isolates and mutants of
Clostridium autoethanogenum include JA1-1 (DSM10061) (Abrini, Arch
Microbiol, 161: 345-351, 1994), LBS1560 (DSM19630) (WO
2009/064200), and LZ1561 (DSM23693). Isolates and mutants of
Clostridium ljungdahlii include ATCC 49587 (Tanner, Int J Syst
Bacteriol, 43: 232-236, 1993), PETCT (DSM13528, ATCC 55383), ERI-2
(ATCC 55380) (U.S. Pat. No. 5,593,886), C-01 (ATCC 55988) (U.S.
Pat. No. 6,368,819), 0-52 (ATCC 55989) (U.S. Pat. No. 6,368,819),
and OTA-1 (Tirado-Acevedo, Production of bioethanol from synthesis
gas using Clostridium ljungdahlii, PhD thesis, North Carolina State
University, 2010). Isolates and mutants of Clostridium ragsdalei
include PI 1 (ATCC BAA-622, ATCC PTA-7826) (WO 2008/028055).
[0131] The substrate generally comprises at least some amount of
CO, such as about 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or
100 mol% CO. The substrate may comprise a range of CO, such as
about 20-80, 30-70, or 40-60 mol% CO. Preferably, the substrate
comprises about 40-70 mol% CO (e.g., steel mill or blast furnace
gas), about 20-30 mol% CO (e.g., basic oxygen furnace gas), or
about 15-45 mol % CO (e.g., syngas). In some embodiments, the
substrate may comprise a relatively low amount of CO, such as about
1-10 or 1-20 mol % CO. The microorganism of the invention typically
converts at least a portion of the CO in the substrate to a
product. In some embodiments, the substrate comprises no or
substantially no (<1 mol %) CO.
[0132] The substrate may comprise some amount of H.sub.2. For
example, the substrate may comprise about 1, 2, 5, 10, 15, 20, or
30 mol % H.sub.2. In some embodiments, the substrate may comprise a
relatively high amount of H.sub.2, such as about 60, 70, 80, or 90
mol % H.sub.2. In further embodiments, the substrate comprises no
or substantially no (<1 mol %) H.sub.2.
[0133] The substrate may comprise some amount of CO.sub.2. For
example, the substrate may comprise about 1-80 or 1-30 mol %
CO.sub.2. In some embodiments, the substrate may comprise less than
about 20, 15, 10, or 5 mol % CO.sub.2. In another embodiment, the
substrate comprises no or substantially no (<1 mol %)
CO.sub.2.
[0134] In certain embodiments, the industrial process is selected
from the group consisting of ferrous metal products manufacturing,
such as a steel mill manufacturing, non-ferrous products
manufacturing, petroleum refining, coal gasification, electric
power production, carbon black production, ammonia production,
methanol production, and coke manufacturing. In these embodiments,
the substrate and/or C1-carbon source may be captured from the
industrial process before it is emitted into the atmosphere, using
any convenient method.
[0135] The substrate and/or C1-carbon source may be syngas, such as
syngas obtained by gasification of coal or refinery residues,
gasification of biomass or lignocellulosic material, or reforming
of natural gas. In another embodiment, the syngas may be obtained
from the gasification of municipal solid waste or industrial solid
waste.
[0136] The composition of the substrate may have a significant
impact on the efficiency and/or cost of the reaction. For example,
the presence of oxygen (O.sub.2) may reduce the efficiency of an
anaerobic fermentation process. Depending on the composition of the
substrate, it may be desirable to treat, scrub, or filter the
substrate to remove any undesired impurities, such as toxins,
undesired components, or dust particles, and/or increase the
concentration of desirable components.
[0137] In certain embodiments, the fermentation is performed in the
absence of carbohydrate substrates, such as sugar, starch, lignin,
cellulose, or hemicellulose.
[0138] "Increasing the efficiency," "increased efficiency," and the
like include, but are not limited to, increasing growth rate,
product production rate or volume, product volume per volume of
substrate consumed, or product selectivity. Efficiency may be
measured relative to the performance of parental microorganism from
which the microorganism of the invention is derived.
[0139] Typically, the culture is performed in a bioreactor. The
term "bioreactor" includes a culture/fermentation device consisting
of one or more vessels, towers, or piping arrangements, such as a
continuous stirred tank reactor (CSTR), immobilized cell reactor
(ICR), trickle bed reactor (TBR), bubble column, gas lift
fermenter, static mixer, or other vessel or other device suitable
for gas-liquid contact. In some embodiments, the bioreactor may
comprise a first growth reactor and a second culture/fermentation
reactor. The substrate may be provided to one or both of these
reactors. As used herein, the terms "culture" and "fermentation"
are used interchangeably. These terms encompass both the growth
phase and product biosynthesis phase of the culture/fermentation
process.
[0140] In certain embodiments, the fermentation is performed in the
absence of light or in the presence of an amount of light
insufficient to meet the energetic requirements of photosynthetic
microorganisms. In certain embodiments, the microorganism of the
invention is a non-photosynthetic microorganism.
[0141] Target products may be separated or purified from a
fermentation broth using any method or combination of methods known
in the art, including, for example, fractional distillation,
evaporation, pervaporation, gas stripping, phase separation, and
extractive fermentation, including for example, liquid-liquid
extraction. In certain embodiments, target products are recovered
from the fermentation broth by continuously removing a portion of
the broth from the bioreactor, separating microbial cells from the
broth (conveniently by filtration), and recovering one or more
target products from the broth. Alcohols and/or acetone may be
recovered, for example, by distillation. Acids may be recovered,
for example, by adsorption on activated charcoal. Separated
microbial cells are preferably returned to the bioreactor. The
cell-free permeate remaining after target products have been
removed is also preferably returned to the bioreactor. Additional
nutrients (such as B vitamins) may be added to the cell-free
permeate to replenish the medium before it is returned to the
bioreactor.
Description
[0142] Energy optimized design for a commercial sized plant. This
design was optimized for a production plant of >50 k ton/year of
ethanol using gas fermentation. It was designed for minimal energy
use and lowest carbon footprint in a location with low
carbon-intensive electricity production.
[0143] The overall distillation plant includes 3 main elements: (i)
a broth stripper at low vacuum conditions with overhead vapor
recompression, (ii) a `rectification` column at atmospheric
conditions with overhead vapor recompression; and (iii) a
dehydration section to bring the ethanol to fuel grade
concentration. This invention focuses on the operation of the broth
stripper at low vacuum conditions.
[0144] The broth stripper section comprises; (i) a broth degasser
at near atmospheric pressure; (ii) a vacuum broth column with
multiple stages of packing; (iii) a vapor recompression package on
the broth column overheads (prefer multiple stage turbofans); (iv)
a reboiler exchanger to transfer heat from the compressed overheads
to the bottom of the column (prefer falling film evaporator); (v) a
trim condenser on the overheads flow after the reboiler exchanger;
and (vi) a return broth cooling tank.
Key Design Features and Reasoning:
[0145] Degasser--a Cyclonic entry degasser at 0-0.5 barg pressure.
This minimizes foaming and fouling risks, which are typically
higher risk in gas fermentation. The Vapor phase is routed to main
gas fermentation plant scrubber to minimize ethanol loss. The
degasser saves on a separate scrubber and vacuum system, or making
the low pressure scrubber and vacuum system much bigger.
[0146] Broth stripper column. A multiple stage packed stripping
column (feed at top) is provided. Structured packing minimizes
liquid residence time for bacteria to less than 5 minutes total.
The multiple stage packed stripping column ideally comprises 8 to
12 theoretical stages to get ethanol <0.2 wt %, or <0.1 wt %.
The stripping column provides a return broth with a low enough
ethanol concentration that a small bleed stream can be taken
without the need to strip further in a separate column.
[0147] Vacuum conditions with a bottom pressure no greater than 90
mbar, 95 mbar, or 100 mbar to ensure bacterial survival.
Temperature throughout the column depends on pressure and
equilibrium ethanol concentration. Pressure drop over the packing
means pressure at the top of the column is lower. This results in a
temperature gradient down the column (low.fwdarw.high), combined
with an ethanol titer gradient (high.fwdarw.low). Since the main
mechanism of cell death due to high temperature and high ethanol
titer are similar and additive (membrane fluidity), this
arrangement minimizes overall stress to the bacteria.
[0148] Liquid distributer designed for high rate of degassing
(still big pressure drop from degas ser with high CO.sub.2 load).
Makes use of large column diameter to lower inlet degassing
requirements.
[0149] Use of a water-only reboiler section with a total trap-out
tray (collecting the broth) above. Broth is not subjected to the
higher skin temperatures of the reboiler exchanger. Broth is not
subjected to the additional residence time of the reboiler level
control volume.
[0150] All vapor to the column is generated from pure water, which
requires makeup. This dilutes the broth return flow. However, since
gas fermentation requires a bleed flow and water makeup, this is
not detrimental.
[0151] Cooling tank on broth return. Rather than pump from a level
control on the total trap-out tray within the column, a system was
devised to have the broth leave the column immediately upon hitting
the tray, and enter a separate vacuum vessel with a liquid seal and
a pump around cooler. Level control volume required for return
broth pump is now at a lower (low stress) temperature.
Significantly reduces overall time at high temperature for the
bacteria
[0152] Mechanical vapor recompression (MVR) on broth stripper.
Compresses overheads vapor, allowing condensing energy to drive
reboiler requirements for the column. Compression ratio required
depends on column bottom temperature (affected mostly by dP over
the packing), and dT required across reboiler exchanger. Main
energy input for the system, so improving efficiencies here really
improves overall energy efficiency (packing dP, reboiler dT).
Eliminates both steam and cooling water energy duty requirements
for the column, in exchange for approximately 1/10 of these duties
as electrical energy in the MVR system.
[0153] Reboiler exchanger--Uses the condensing energy of the
compressed overhead vapor stream to reboil the water section at the
bottom of the column. Preferred to use falling film evaporator to
minimize liquid head associated with other exchanger types. Even
relatively small heights of liquid head in the exchanger would
increase pressure and therefore required boiling temperature on the
column bottom side of the exchanger, which would increase required
output pressure of the MVR (or massively increase required
exchanger area).
[0154] Trim condenser and vacuum scrubber--To minimize vapor flow
to vacuum pump system and capture as much ethanol as possible.
Standard equipment.
[0155] Rectification column--Standard multistage column for
splitting ethanol and water. Preferred to use packing combined with
MVR system for highest efficiency. Traditional steam driven
reboiler and cooling water condenser systems are also possible. In
this case energy integration is possible for overheads: condensing
energy can be used to drive the broth stripper reboiler which
results in smaller MVR and reboiler exchanger requirements, but
increases the size of the trim condenser.
[0156] Dehydration system--Membrane dehydration is preferred due to
more options on heat integration. Mol sieve technology also
possible.
[0157] Water integration--The water from the bottom of the
rectifier can be recycled directly back to feed the water-only
reboiler area at the bottom of the vacuum broth stripper. This is a
combination of water originally stripped in the vacuum broth
stripper, and water added in the vacuum scrubber. For gas
fermentation, this will include some organic acids that are
stripped in the broth stripper, but they will also vaporize again
at the bottom of the broth stripper, so there will be no (or
minimal) concentration factor
[0158] Vacuum distillation has been found to effectively recover
product from fermentation broth while ensuring the viability of the
microorganisms contained in the fermentation broth. The
fermentation broth being fed to the vacuum distillation vessel is
sourced from a bioreactor. Preferably, the bioreactor is used for
fermentation of a C1-containing gaseous substrate. In order for the
fermentation process to operate continuously, at least a portion of
the microorganisms contained in the broth must remain viable. These
microorganisms have fairly specific tolerances to concentrations of
certain products. Additionally, these microorganisms have fairly
specific tolerances to temperature. For example, in at least one
embodiment, the microorganisms have an optimum growth temperature
of 37.degree. C. The inventors have found that by utilizing vacuum
distillation, the conditions for viability are able to be
controlled in such a manner that continuous operation of the
fermentation process is possible.
[0159] The vacuum distillation vessel consists of multiple
elements: (1) an exterior casing defining at least one inlet for
receiving fermentation broth, one outlet for transferring a product
enriched stream, and one outlet for transferring a product depleted
stream; (2) a separation section located within the casing, the
separation section being bounded above by an upper tray and below
by a lower tray, the separation section defining a separation
medium for providing a plurality of theoretical distillation
stages; and (3) a liquid level maintained at the bottom of the
vacuum distillation vessel.
[0160] The vacuum distillation vessel is coupled with the
bioreactor so as to effectively process the fermentation broth. It
was found by the inventors that by feeding the vacuum distillation
vessel at a given feed rate, product accumulation in the bioreactor
is controlled, thereby ensuring the viability of the
microorganisms. Feed rate is given in terms of volumes of
fermentation broth of the bioreactor per hour. The inventors have
identified that a feed rate between 0.05 and 0.5 reactor volumes
per hour allows for the broth to be effectively processed, while
ensuring the viability of the microorganisms. The feed rate may be
dependent, at least in part, on the vacuum distillation vessel
conditions, including but not limited to, pressure, temperature,
residence time, product concentration in fermentation broth, steam
feed rate, and/or separation medium. In certain embodiments, the
feed rate is between 0.05 to 0.1, 0.05 to 0.2, 0.05 to 0.3, 0.05 to
0.4, 0.1 to 0.3, 0.1 to 0.1 to 0.5, or 0.3 to 0.5 reactor volumes
per hour. Preferably, the feed rate is controlled such that the
product depleted stream has acceptable proportions of product.
[0161] Additionally, the inventors have identified that by keeping
the residence time, being defined as the time that the fermentation
broth is within the vacuum distillation vessel, within a certain
period of time, the viability of the microorganisms is ensured. The
inventors have identified that a residence time between 0.5 and 15
minutes allows for the broth to be effectively processed, while
ensuring the viability of the microorganisms. In various
embodiments, the residence time is between 0.5 and 12 minutes, 0.5
and 9 minutes, 0.5 and 6 minutes, 0.5 and 3 minutes, 2 and 15
minutes, 2 and 12 minutes, 2 and 9 minutes, or 2 and 6 minutes. In
at least one embodiment, the residence time is less than 15
minutes, less than 12 minutes, less than 9 minutes, less than 6
minutes, less than 3 minutes, less than 2 minutes, or less than 1
minute to ensure the viability of the microorganisms.
[0162] The vacuum distillation vessel processes the fermentation
broth through use of pressure reduction, where the pressure inside
the vacuum distillation vessel is maintained below atmospheric so
as to lower the temperature necessary to vaporize the liquid in the
fermentation broth. The temperature in the vacuum distillation
vessel may be dependent on the pressure and ethanol concentration.
Preferably, the liquid being vaporized is primarily product, such
as ethanol. Preferably, the pressure of the vacuum distillation
vessel is maintained between 40 mbar(a) and 100 mbar(a) to ensure
the viability of the microorganisms. In at least one embodiment,
the vacuum distillation vessel is maintained between 40 mbar(a) and
80 mbar(a), between 40 mbar(a) and 90 mbar(a), or between 45
mbar(a) to 90 mbar(a). The pressure typically drops over the
separation medium, meaning that the pressure at the top of the
vacuum distillation vessel is lower relative to the pressure at the
bottom of the vacuum distillation vessel. Preferably, the pressure
drop over the height of the vacuum distillation vessel is less than
32 mbar. In certain instances, the pressure drop over the height of
the vacuum distillation vessel is less than 30 mbar, less than 28
mbar, less than 26 mbar, less than 24 mbar, less than 22 mbar, less
than 20 mbar, or less than 18 mbar.
[0163] This results in a temperature gradient within the vacuum
distillation vessel where the temperature increases over the length
of the vessel, being lowest at the top of the vacuum distillation
vessel and highest at the bottom of the vacuum distillation vessel.
As the fermentation broth flows down the vacuum distillation vessel
the product titer is reduced, where the product titer is highest at
the top of the vacuum distillation vessel and lowest at the bottom
of the vacuum distillation vessel.
[0164] The fermentation broth initially enters the vacuum
distillation vessel via an inlet in the casing. The inlet for
receiving the fermentation broth is located above the upper tray.
As the fermentation broth enters the vessel, a portion of the
product in the fermentation broth is vaporized forming a product
enriched stream, which rises toward the top of the vessel, exiting
through an outlet in the casing. The outlet for transferring the
product enriched stream is elevated relative to the inlet for
receiving the fermentation broth. The remaining fermentation broth
passes through the upper tray and through the separation medium.
The separation medium provides a plurality of theoretical
distillation stages. As the fermentation broth reaches each
theoretical distillation stage additional product is vaporized. The
vaporized product becoming part of the product enriched stream,
rising toward the top of the vessel, and exiting through an outlet
in the casing. After passing through the separation medium, the
remaining fermentation broth exits the vacuum distillation vessel
via an outlet in the casing. The fermentation broth exiting the
casing is the product depleted stream. The product depleted stream
contains viable microbial biomass. The outlet for transferring the
product depleted stream is elevated relative to the lower tray. The
lower tray is elevated relative to the bottom of the vacuum
distillation vessel. The bottom of the vacuum distillation vessel
contains a level of liquid.
[0165] In order to increase the effectiveness of the vacuum
distillation vessel and provide for the necessary vapor-liquid
contact, a vapor stream may be introduced from a reboiler to the
vacuum distillation vessel via an inlet in the casing. The inlet
for receiving the vapor stream is located subjacent to the lower
tray. The reboiler utilizes a portion of the liquid from the bottom
of the vacuum distillation vessel in combination with energy to
vaporize the liquid and create the vapor stream. The liquid from
the bottom of the vacuum distillation vessel is transferred via an
outlet in the vacuum distillation vessel casing. This outlet is
located lower than the inlet for receiving the vapor stream. The
vapor stream flows upward through the separation medium, picks up
portions of product, and becomes part of the product enriched
stream. The product enriched stream exiting through the outlet for
transferring the product enriched stream. In one or more
embodiment, the product enriched stream may be further processed in
order to increase the concentration of the product.
[0166] The fermentation broth being passed to the vacuum
distillation vessel may contain proportions of gas. Gas in the
fermentation broth may result in a decrease in performance of the
vacuum distillation vessel. To prevent the performance decrease
associated with gas in the fermentation broth, a degassing vessel
may be utilized. Preferably, the degassing vessel is a cyclonic
degasser. Preferably, the degassing vessel is operated at a
pressure between 0.0 bar(g) and 1.0 bar(g). In one embodiment, the
degassing vessel is operated at a pressure between 0.0 bar(g) and
0.5 bar(g). Preferably, the degassing vessel removes substantially
all of the gas from the fermentation broth. In particular
embodiments, the degassing vessel removes between 0 and 100% of the
gas in the fermentation broth. In certain instances, the degassing
vessel removes more than 20%, more than 40%, more than 60%, or more
than 80% of the gas from the fermentation broth. The degassing
vessel is operated so as to separate at least a portion of the gas
from the fermentation broth. When utilizing a cyclonic degasser,
the fermentation broth is rotated, creating a low-pressure region
at the center of the rotating fermentation broth, causing the gas
to separate from the fermentation broth. The fermentation broth
with reduced proportions of gas is then sent to the vacuum
distillation vessel. The separated gas may contain proportions of
product. To recover product from the separated gas and avoid loss
of product, the separated gas may be sent to a subsequent device
and/or processing. In at least one embodiment, the separated gas
may be passed to the bioreactor.
[0167] Preferably, the product depleted stream leaving the vacuum
distillation vessel is passed back to the bioreactor. The product
depleted stream contains viable microbial biomass, which, if passed
back to the bioreactor, will increase the efficiency of the
fermentation process. However, this product depleted stream may
have a higher than optimal temperature. Therefore, prior to being
passed back to the bioreactor, the product depleted stream may
undergo cooling. The cooling of the product depleted stream may be
completed by way of a cooling means. The cooling is conducted under
conditions to reduce the temperature of the product depleted stream
such that the product depleted stream temperature is within an
optimal range. By reducing the temperature of the product depleted
stream prior to passing the product depleted stream to the
bioreactor, unnecessary heating of the culture in the bioreactor
can be avoided. For example, if the product depleted stream were to
be provided to the bioreactor at a higher temperature relative to
the fermentation broth within the bioreactor, then the recycling of
the product depleted stream could result in a temperature increase
of the fermentation broth within the bioreactor. If the temperature
of the fermentation broth within the bioreactor is not maintained
within an acceptable range, suitable for the microorganisms, then
the viability of the microorganisms could decrease. Thus,
monitoring and controlling the temperature of the product depleted
stream may be critical to the ability of recycling the product
depleted stream.
[0168] FIG. 1 shows a vacuum distillation vessel 100 for recovering
at least one product from a fermentation broth, the fermentation
broth being delivered from a bioreactor. The vacuum distillation
vessel 100 comprises an exterior casing 113, defining an inlet 114
for receiving fermentation broth, an outlet 115 for transferring a
product enriched stream via piping 104, and an outlet 116 for
transferring a product depleted stream. The vacuum distillation
vessel 100 also comprises a separation section 109 located within
the casing 113, the separation section 109 is bounded above by an
upper tray 112 and below by a lower tray 111. The vacuum
distillation vessel 100 is designed in a way to increase the
recovery of product from the fermentation broth. The outlet 115 for
transferring the product enriched stream is elevated relative to
the inlet 114 for receiving the fermentation broth. The inlet 114
for receiving the fermentation broth being elevated relative to the
upper tray 112, the outlet 116 for transferring the product
depleted stream being elevated relative to the lower tray 111.
[0169] The vacuum distillation vessel 100 is designed such that the
vacuum distillation vessel 100 can process fermentation broth at a
given feed rate. The feed rate is defined in terms of volume of
fermentation broth in the bioreactor. Preferably, the vacuum
distillation vessel 100 is designed such that the feed rate is
between 0.05 to 0.5.
[0170] The vacuum distillation vessel 100 is designed such that the
fermentation broth defines a residence time. The residence time is
defined in terms of the amount of time the fermentation broth is
within the vacuum distillation vessel 100. The fermentation broth
is deemed to be within the vacuum distillation vessel 100 when the
fermentation broth enters through the inlet 114. The fermentation
broth is deemed to be out of the vacuum distillation vessel 100
when the fermentation broth exits through the outlet 116.
Preferably, the residence time is between 0.5 and 15 minutes. In
various embodiments, the residence time is between 0.5 and 12
minutes, 0.5 and 9 minutes, 0.5 and 6 minutes, 0.5 and 3 minutes, 2
and 15 minutes, 2 and 12 minutes, 2 and 9 minutes, or 2 and 6
minutes. In at least one embodiment, the residence time is less
than 15 minutes, less than 12 minutes, less than 9 minutes, less
than 6 minutes, less than 3 minutes, less than 2 minutes, or less
than 1 minute to ensure the viability of the microorganisms.
[0171] The given residence time may depend, at least in part, on
the type of separation medium 109 within the vacuum distillation
vessel 100. In at least one embodiment, the separation medium 109
is defined by a series of distillation trays. Preferably, a
separation medium 109 is provided such that a sufficient number of
theoretical distillation stages are provided to recover product.
Preferably, the separation medium 109 provides multiple theoretical
distillation stages. In other embodiments, the separation medium
109 provides a minimum number of theoretical distillation stages,
for example, more than 3 theoretical distillation stages, more than
4 theoretical distillation stages, more than 5 theoretical
distillation stages, or more than 6 theoretical distillation
stages.
[0172] The vacuum distillation vessel 100 is designed so as to
effectively recover product in the fermentation broth and prevent
product accumulation in the bioreactor. Preferably, the product
depleted stream has reduced proportions of product such that
product accumulation is effectively reduced or eliminated. In at
least one embodiment, the product depleted stream comprises less
than 0.2 wt. % product. In certain embodiments, the product
depleted stream comprises less than 1.0 wt. % product. In
particular instances, the product depleted stream comprises between
0.1 and 1.0 wt. % product. In at least one embodiment, the product
being recovered is ethanol.
[0173] To effectuate the transfer of the product depleted stream,
the outlet 116 for transferring the product depleted stream may be
connected via piping means 102 to the bioreactor. The product
depleted stream may have higher than acceptable temperature, and
thus may require cooling prior to being transferred to the
bioreactor. To effectuate cooling, a cooling means may be provided.
The cooling means may bring the product depleted stream to an
acceptable temperature prior to the product depleted stream being
transferred to the bioreactor.
[0174] In some instances, the fermentation broth may have higher
than acceptable proportions of gas, and thus may require degassing
prior to being transferred to the bioreactor. To effectuate
degassing, a degassing vessel 200 may be provided. Preferably, the
degassing vessel 200 is a cyclonic degas ser. The degassing vessel
200 may comprise an inlet 201 for receiving the fermentation broth.
This inlet 201 may be connected via piping means 702 to the
bioreactor in order to transfer the fermentation broth from the
bioreactor. Preferably, the degassing vessel 200 is operated such
that at least a portion of gas can be removed from the fermentation
broth prior to the fermentation broth being delivered to the vacuum
distillation vessel 100. The degassing vessel 200 is capable of
separating the gas from the fermentation broth such the
fermentation broth is separated into an evolved gas stream and a
degassed fermentation broth. The evolved gas stream exits the
degassing vessel 200 via the outlet 205. The outlet 205 may be
connected via piping means 204 to a subsequent process to recover
product from the evolved stream. In at least one embodiment, the
evolved gas stream is water scrubbed to recover product in the
evolved gas stream. Additionally, the outlet 205 may be connected
to the bioreactor via piping means 204 where the evolved gas may be
used in the fermentation process. The degassed fermentation broth
is passed through an outlet 203 to the vacuum distillation vessel
100 via piping means 202. In at least one embodiment, the degassing
vessel 200 is operated at a pressure between 0.0 bar(g) and 0.5
bar(g). In embodiments not utilizing a degassing vessel 200, the
fermentation broth may be sent directly from the bioreactor to the
inlet 114 in the vacuum distillation vessel 100 via piping means
702.
[0175] The vacuum distillation vessel 100 is designed so as to
ensure the viability of the microorganisms while providing product
recovery. Preferably, the viability of the microorganisms in the
product depleted stream is greater than 85 percent. In at least one
embodiment, the viability of the microorganisms in the product
depleted stream is substantially equal to the viable microbial
biomass in the incoming fermentation broth.
[0176] The vacuum distillation vessel 100 may provide for product
recovery through use of a reboiler 800. The reboiler 800 is
provided so as to direct a vapor stream to the vacuum distillation
vessel 100. This vapor stream is directed through piping means 802
from the outlet 806 in the reboiler to the inlet 117 in the casing
113 of the vacuum distillation vessel 100. The vapor stream enters
the vacuum distillation vessel 100 and rises upward through the
lower plate 111 and the separation medium 109 contacting the
product in the fermentation broth. The reboiler 800 may create the
vapor stream through use of liquid 107 located in the bottom of the
vacuum distillation vessel 100. Preferably, this liquid 107 is
comprised substantially of water and minimal amounts of microbial
biomass. The liquid 107 may be passed through piping means 106 from
an outlet 118 in the vacuum distillation vessel 100 to an inlet 801
in the reboiler 800. In various embodiments, the liquid 107 located
in the bottom of the vacuum distillation vessel 100 may be derived
from a number of sources including, but not limited to, the cooling
means, steam condensate, a cogeneration unit, and/or the
rectification column bottoms.
[0177] The casing 113 of the vacuum distillation vessel 100 may
comprise one or more additional inlets 121, 119 and outlet 120 for
transferring liquid 107 via piping 101, 103, and 105 into and out
of the vacuum distillation vessel 100. This may allow for the
content and proportion of the liquid 107 in the vacuum distillation
vessel 100 to be controlled. In certain instances, the piping 101,
103, and 105 may be connected to one or more of the sources of the
liquid 107.
[0178] Additionally, the vacuum distillation vessel 100 may be
designed such that the vacuum distillation vessel 100 is separated
into multiple compartments in a manner where fermentation broth
from multiple bioreactors may be passed to the vacuum distillation
vessel 100 without mixing. This separation may be achieved through
any means suitable to ensure such separation.
[0179] The vacuum distillation vessel may contain one or more
additional tray 122 below the lower tray 111. FIG. 2 illustrates a
vacuum distillation vessel 100 with additional trays 122 below the
lower tray 111. These additional trays 122 provide for additional
product removal. The vacuum distillation vessel 100 is designed to
transfer fermentation broth, containing the viable microbial
biomass, to the bioreactor through the outlet 116, which is placed
above the lower tray 111. The fermentation broth that passes
through the lower tray 111 may contain additional, albeit minimal,
amounts of fermentation broth containing the viable microbial
biomass. The fermentation broth that passes through the lower tray
111 is not passed to the bioreactor. This fermentation broth is
instead passed through the one or more additional trays 122 where
additional product is recovered from the fermentation broth. After
passing through the one or more additional trays 122, the
fermentation broth mixes with the liquid 107 located in the bottom
of the vacuum distillation vessel 100. This liquid 107, including
portions of fermentation broth containing microbial biomass, is
then passed to the reboiler 800 to produce the vapor stream.
[0180] FIGS. 3 and 4 illustrate the need for a vacuum distillation
vessel to remove product from the fermentation broth. FIG. 3 shows
the metabolite profile of a batch fermentation run. FIG. 3 shows
that the biomass and ethanol concentration increases exponentially
during the initial phase of the fermentation run. As the ethanol
accumulates, exceeding a concentration around 30 g/L, the biomass
slows down due to the effects of the ethanol on the microbes. This
is further shown by FIG. 4, where the CO uptake and CO.sub.2
production slows down around the same time that the ethanol
concentration reaches around 30 g/L. This data illustrates the
needs for the vacuum distillation vessel of the current invention,
where product concentration rates can be controlled to the point
where the negative effects of product accumulation are mitigated
and/or reduced.
[0181] The vacuum distillation vessel is capable of recycling
product depleted fermentation broth to the bioreactor. The vacuum
distillation vessel is designed to recover products, while ensuring
the viability of the microorganisms so that, when recycled, the
microorganisms may ferment the C1-containing gas in the bioreactor
to produce products. FIGS. 5 and 6 illustrate the ability of the
vacuum distillation vessel to ensure the viability of the
microorganisms from multiple variations of bioreactor designs.
[0182] FIG. 5 shows the viability of microorganisms from a
bioreactor with a certain configuration, where the fermentation
broth is recycled from the vacuum distillation vessel to the
bioreactor. The viability of the microorganisms was measured at
three times intervals from the bioreactor (Bioreactor 1) and from
the vacuum distillation vessel (VD return). As is shown in the
graph, the viability of the microorganisms in the vacuum
distillation vessel is substantially equal to the viability of the
microorganisms in the bioreactor.
[0183] FIG. 6 shows the viability of the microorganisms from a
bioreactor with a different configuration, where the fermentation
broth is recycled from the vacuum distillation vessel to the
bioreactor. The viability of the microorganisms was measured at
three times intervals from the bioreactor (Bioreactor 2) and from
the vacuum distillation vessel (VD return). As shown in the graph,
the viability of the microorganisms in the vacuum distillation
vessel is substantially equal to the viability of the
microorganisms in the bioreactor.
[0184] FIG. 7 is a diagram showing the system according to one
embodiment of the invention. The system is an energy optimized
design 700 for a commercial sized plant, with minimal energy use
and lowest carbon footprint. Broth 702 comprising product from a
fermentation process is passed to degasser 704. The product
exemplified is ethanol. Degasser 704 may be a cyclonic entry
atmospheric degasser. Vapor phase 706 may be routed to a scrubber
of the fermentation process (not shown). Degassed broth 708 is
passed to separation vessel 710 at a location near to the top of
the vessel. Separation vessel 710 maybe a stripping column and may
be a vacuum stripper as described above. Product depleted broth 712
is removed from vessel 710 at a location proximate to a total
trap-out tray of vessel 710 and is passed to cooling tank 714.
Cooling water in stream 716 is indirectly heat exchanged in heat
exchanger 718 to cool a portion of product depleted broth 720
removed from cooling tank 714 generating cooled stream 719 which is
passed to cooling tank 714. Another portion of product depleted
broth 720 removed from cooling tank 714 is passed to bioreactors of
the fermentation process (not shown). A bleed stream 722 is removed
from product depleted broth 720 removed from cooling tank 714 and
may be passed to wwtp (wastewater treatment plant) not shown.
Boiler feed water 724 is passed into the bottom of vessel 710 and
purge stream 726 is removed from the bottom of vessel 710. Liquid
water stream 728 is also removed from the bottom of vessel 710 and
passed to reboiler 730 where liquid water stream is heated and
returned to vessel 710.
[0185] Product rich vapor stream 732 is removed as overhead from
vessel 710. Product rich vapor stream 732 is compressed in
multistage mechanical vapor recompression unit 734 to generate
compressed stream 736. Compressed stream 734 is passed to reboiler
730 and at least partially condensed thereby providing energy to
drive reboiler 730 and heat liquid water stream 728. Partially
condensed stream 738 is passed to trim condenser 740 and heat
exchanged with cooling water stream 742. Non-condensed components
removed in stream 744 and passed along with water stream 748 to
vacuum scrubber 746 where remaining product ethanol is scrubbed
into stream 752. Non-condensable components not scrubbed into the
process water are removed as scrubber overhead stream 750. Trim
condenser 740 provides condensed stream comprising ethanol 754
which is combined with stream 752 from scrubber 746 to form
combined stream 756 which in turn is passed to rectification column
760. In rectification column 760, product ethanol is distilled into
overhead 762. Overhead 762 is compressed in compressed in
multistage mechanical vapor recompression unit 766 to generate
compressed stream 768 which is passed to heat exchanger 770 to heat
rectification column liquid reboil stream 772. After heat exchange,
stream 768 is passed to dehydration system 774 to generate dry
product stream 776. Optionally a portion of stream 768 may be
passed back to rectification column in stream 778 as reflux. Water
stream 780 is removed from the bottom of rectification column 760
and recycled to the bottom of vessel 710. Through this recycle,
water stream 780 also directly provides heat to the bottom of
vessel 710. Water stream 780 may be indirectly heat exchanged with
stream 756 in heat exchanger 758 to heat stream 756.
[0186] All references, including publications, patent applications,
and patents, cited herein are hereby incorporated by reference to
the same extent as if each reference were individually and
specifically indicated to be incorporated by reference and were set
forth in its entirety herein. The reference to any prior art in
this specification is not, and should not be taken as, an
acknowledgement that that prior art forms part of the common
general knowledge in the field of endeavour in any country.
[0187] The use of the terms "a" and "an" and "the" and similar
referents in the context of describing the invention (especially in
the context of the following claims) are to be construed to cover
both the singular and the plural, unless otherwise indicated herein
or clearly contradicted by context. The terms "comprising,"
"having," "including," and "containing" are to be construed as
open-ended terms (i.e., meaning "including, but not limited to")
unless otherwise noted. Recitation of ranges of values herein are
merely intended to serve as a shorthand method of referring
individually to each separate value falling within the range,
unless otherwise indicated herein, and each separate value is
incorporated into the specification as if it were individually
recited herein. All methods described herein can be performed in
any suitable order unless otherwise indicated herein or otherwise
clearly contradicted by context. The use of any and all examples,
or exemplary language (e.g., "such as") provided herein, is
intended merely to better illuminate the invention and does not
pose a limitation on the scope of the invention unless otherwise
claimed. No language in the specification should be construed as
indicating any non-claimed element as essential to the practice of
the invention.
[0188] Preferred embodiments of this invention are described
herein. Variations of those preferred embodiments may become
apparent to those of ordinary skill in the art upon reading the
foregoing description. The inventors expect skilled artisans to
employ such variations as appropriate, and the inventors intend for
the invention to be practiced otherwise than as specifically
described herein. Accordingly, this invention includes all
modifications and equivalents of the subject matter recited in the
claims appended hereto as permitted by applicable law. Moreover,
any combination of the above-described elements in all possible
variations thereof is encompassed by the invention unless otherwise
indicated herein or otherwise clearly contradicted by context.
* * * * *