U.S. patent application number 13/060334 was filed with the patent office on 2011-11-17 for methods of sustaining culture viability.
This patent application is currently assigned to LANZATECH NEW ZEALAND LIMITED. Invention is credited to Bakir Al-Sinawi, Christophe Collet, Sean Dennis Simpson.
Application Number | 20110281336 13/060334 |
Document ID | / |
Family ID | 42665729 |
Filed Date | 2011-11-17 |
United States Patent
Application |
20110281336 |
Kind Code |
A1 |
Simpson; Sean Dennis ; et
al. |
November 17, 2011 |
METHODS OF SUSTAINING CULTURE VIABILITY
Abstract
The present invention relates to methods for sustaining a
microbial culture during periods of limited substrate supply. In
accordance with the methods of the invention a microbial culture
comprising carboxydotrophic bacteria can be sustained during
periods of limited substrate supply by maintaining the temperature
of the microbial culture at a temperature below an optimum
operating temperature.
Inventors: |
Simpson; Sean Dennis;
(Parnell, NZ) ; Collet; Christophe; (Auckland,
NZ) ; Al-Sinawi; Bakir; (Auckland, NZ) |
Assignee: |
LANZATECH NEW ZEALAND
LIMITED
Parnell
NZ
|
Family ID: |
42665729 |
Appl. No.: |
13/060334 |
Filed: |
February 23, 2010 |
PCT Filed: |
February 23, 2010 |
PCT NO: |
PCT/NZ10/00029 |
371 Date: |
August 3, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61155870 |
Feb 26, 2009 |
|
|
|
Current U.S.
Class: |
435/252.7 ;
435/252.1; 435/286.1 |
Current CPC
Class: |
Y02E 50/17 20130101;
C12P 7/40 20130101; C12N 1/04 20130101; C12P 7/065 20130101; C12N
1/20 20130101; Y02E 50/10 20130101; C12P 7/06 20130101; C12P 7/16
20130101; C12Q 3/00 20130101 |
Class at
Publication: |
435/252.7 ;
435/252.1; 435/286.1 |
International
Class: |
C12N 1/20 20060101
C12N001/20; C12M 1/36 20060101 C12M001/36 |
Claims
1-24. (canceled)
25. A method of sustaining viability of a microbial culture of
carboxydotrophic bacteria when a substrate that includes CO is
limited or unavailable, said method comprising maintaining the
culture substantially at a temperature or within a temperature
range below an optimum operating temperature of the culture.
26. The method of claim 25, wherein maintaining the culture
comprises maintaining the temperature at least 5.degree. C. below
the optimum operating temperature.
27. The method of claim 25, further comprising maintaining the
temperature of the microbial culture substantially at the optimum
operating temperature if the substrate comprising CO becomes
non-limited.
28. The method of claim 25, wherein the microbial culture is in
liquid nutrient media in a bioreactor.
29. The method of claim 28, further comprising cooling the
bioreactor such that the temperature of the liquid nutrient media
is maintained at a temperature below the optimum operating
temperature.
30. The method of claim 25, further comprising sustaining viability
of the culture over a period of limited CO availability of at least
3 hours.
31. The method of claim 25, wherein the carboxydotrophic bacteria
is selected from the group consisting of Clostridium, Moorella,
Pyrococcus, Eubacterium, Desulfobacterium, Carboxydothermus,
Acetogenium, Acetobacterium, Acetoanaerobium, Butyribaceterium and
Peptostreptococcus.
32. The method of claim 25, wherein the carboxydotrophic bacteria
is Clostridium autoethanogenum.
33. The method of claim 25, wherein the substrate comprises a gas
obtained as a by-product of an industrial process selected from the
group consisting of ferrous metal products manufacturing,
non-ferrous products manufacturing, petroleum refining processes,
gasification of biomass, gasification of coal, electric power
production, carbon black production, ammonia production, methanol
production and coke manufacturing.
34. The method of claim 25, wherein the substrate that includes CO
comprises at least about 15% to about 100% CO by volume.
35. A method of sustaining viability of a microbial culture of
carboxydotrophic bacteria during storage, said method comprising:
cooling the microbial culture to a temperature or temperature range
below an optimum operating temperature; and storing the microbial
culture under limited CO conditions for a selected period of
time.
36. The method of claim 35, further comprising returning the
culture to the optimum operating temperature under non-limited CO
conditions.
37. The method of claim 35, wherein storing the microbial culture
comprises storing the microbial culture for at least 5 hours.
38. The method of claim 37, further comprising transporting the
microbial culture to a remote location during storage.
39. The method of claim 37, further comprising inoculating the
bioreactor with the microbial culture following storage.
40. The method of claim 35, wherein the carboxydotrophic bacteria
are selected from the group consisting of Clostridium, Moorella,
Pyrococcus, Eubacterium, Desulfobacterium, Carboxydothermus,
Acetogenium, Acetobacterium, Acetoanaerobium, Butyribaceterium and
Peptostreptococcus.
41. The method of claim 35, wherein the carboxydotrophic bacteria
is Clostridium autoethanogenum.
42. A system for fermentation of a substrate comprising CO, said
system comprising: at least one bioreactor; determining means
adapted to determine whether a supply of a CO-containing substrate
that is provided to a microbial culture is limited or non-limited;
and temperature control means configured such that, in use, the
temperature of the bioreactor can be adjusted in response to
determination of whether the supply of the CO-containing substrate
for the microbial culture is limited or non-limited.
43. The system of claim 42, wherein the temperature control means
is configured to reduce the temperature of the bioreactor if the
determining means determines that the supply of the CO-containing
substrate is limited.
44. The system of claim 43, wherein the system further comprises
processing means configured such that the temperature of the
bioreactor can be automatically regulated in response to changes in
whether the supply of the CO-containing substrate is limited or
non-limited.
Description
FIELD OF THE INVENTION
[0001] This invention relates generally to methods for increasing
the efficiency of microbial growth and production of products by
microbial fermentation on gaseous substrates. More particularly the
invention relates to processes for producing products such as
alcohols by microbial fermentation before, during and/or after a
substrate stream comprising CO becomes limited. In particular
embodiments, the invention relates to methods of sustaining
viability of a microbial culture during periods of limited
substrate comprising CO.
BACKGROUND OF THE INVENTION
[0002] Ethanol is rapidly becoming a major hydrogen-rich liquid
transport fuel around the world. Worldwide consumption of ethanol
in 2005 was an estimated 12.2 billion gallons. The global market
for the fuel ethanol industry has also been predicted to continue
to grow sharply in future, due to an increased interest in ethanol
in Europe, Japan, the USA and several developing nations.
[0003] For example, in the USA, ethanol is used to produce E10, a
10% mixture of ethanol in gasoline. In E10 blends, the ethanol
component acts as an oxygenating agent, improving the efficiency of
combustion and reducing the production of air pollutants. In
Brazil, ethanol satisfies approximately 30% of the transport fuel
demand, as both an oxygenating agent blended in gasoline, and as a
pure fuel in its own right. Also, in Europe, environmental concerns
surrounding the consequences of Green House Gas (GHG) emissions
have been the stimulus for the European Union (EU) to set member
nations a mandated target for the consumption of sustainable
transport fuels such as biomass derived ethanol.
[0004] The vast majority of fuel ethanol is produced via
traditional yeast-based fermentation processes that use crop
derived carbohydrates, such as sucrose extracted from sugarcane or
starch extracted from grain crops, as the main carbon source.
However, the cost of these carbohydrate feed stocks is influenced
by their value as human food or animal feed, and the cultivation of
starch or sucrose-producing crops for ethanol production is not
economically sustainable in all geographies. Therefore, it is of
interest to develop technologies to convert lower cost and/or more
abundant carbon resources into fuel ethanol.
[0005] CO is a major, free, energy-rich by-product of the
incomplete combustion of organic materials such as coal or oil and
oil derived products. For example, the steel industry in Australia
is reported to produce and release into the atmosphere over 500,000
tonnes of CO annually.
[0006] Catalytic processes may be used to convert gases consisting
primarily of CO and/or CO and hydrogen (H.sub.2) into a variety of
fuels and chemicals. Micro-organisms may also be used to convert
these gases into fuels and chemicals. These biological processes,
although generally slower than chemical reactions, have several
advantages over catalytic processes, including higher specificity,
higher yields, lower energy costs and greater resistance to
poisoning.
[0007] The ability of micro-organisms to grow on CO as a sole
carbon source was first discovered in 1903. This was later
determined to be a property of organisms that use the acetyl
coenzyme A (acetyl CoA) biochemical pathway of autotrophic growth
(also known as the Woods-Ljungdahl pathway and the carbon monoxide
dehydrogenase/acetyl CoA synthase (CODH/ACS) pathway). A large
number of anaerobic organisms including carboxydotrophic,
photosynthetic, methanogenic and acetogenic organisms have been
shown to metabolize CO to various end products, namely CO.sub.2,
H.sub.2, methane, n-butanol, acetate and ethanol. While using CO as
the sole carbon source, all such organisms produce at least two of
these end products.
[0008] Anaerobic bacteria, such as those from the genus
Clostridium, have been demonstrated to produce ethanol from CO,
CO.sub.2 and H.sub.2 via the acetyl CoA biochemical pathway. For
example, various strains of Clostridium Ijungdahlii that produce
ethanol from gases are described in WO 00/68407, EP 117309, U.S.
Pat. Nos. 5,173,429, 5,593,886, and 6,368,819, WO 98/00558 and WO
02/08438. The bacterium Clostridium autoethanogenum sp is also
known to produce ethanol from gases (Abrini et al., Archives of
Microbiology 161, pp 345-351 (1994)).
[0009] However, ethanol production by micro-organisms by
fermentation of gases is always associated with co-production of
acetate and/or acetic acid. As some of the available carbon is
converted into acetate/acetic acid rather than ethanol, the
efficiency of production of ethanol using such fermentation
processes may be less than desirable. Also, unless the
acetate/acetic acid by-product can be used for some other purpose,
it may pose a waste disposal problem. Acetate/acetic acid is
converted to methane by micro-organisms and therefore has the
potential to contribute to GHG emissions.
[0010] Several enzymes known to be associated with the ability of
micro-organisms to use carbon monoxide as their sole source of
carbon and energy are known to require metal co-factors for their
activity. Examples of key enzymes requiring metal cofactor binding
for activity include carbon monoxide dehydrogenase (CODH), and
acetyl --CoA synthase (ACS).
[0011] WO2007/117157 and WO2008/115080, the disclosure of which are
incorporated herein by reference, describe processes that produce
alcohols, particularly ethanol, by anaerobic fermentation of gases
containing carbon monoxide. Acetate produced as a by-product of the
fermentation process described in WO2007/117157 is converted into
hydrogen gas and carbon dioxide gas, either or both of which may be
used in the anaerobic fermentation process.
[0012] The fermentation of gaseous substrates comprising CO, to
produce products such as acids and alcohols, typically favours acid
production. Alcohol productivity can be enhanced by methods known
in the art, such as methods described in WO2007/117157,
WO2008/115080, WO2009/022925 and WO2009/064200, which are fully
incorporated herein by reference.
[0013] In order to sustain viability of one or more
carboxydotrophic bacteria, such as acetogenic bacteria, a
substantially continuous substrate stream comprising sufficient
quantities of CO must be made available to the microbial culture.
Accordingly, if a sufficient amount of CO (or CO2/H2) is not made
available to the microbial culture, the culture may deteriorate and
ultimately die. For example during times of insufficient CO supply,
such as periods of storage, limited substrate supply or
culture/inoculum transfer, a microbial culture will rapidly deplete
the available CO and viability will deteriorate.
[0014] WO2009/114127 provides a method of sustaining viability of
microorganisms during periods of limited substrate supply. However,
the method includes adding CO2 to the bioreactor wherein a
significant amount of ethanol is converted into acetate, resulting
in a decrease in pH. This effect needs to be counteracted to
prevent inhibition by excess molecular acetic acid.
[0015] It is an object of the present invention to provide a
process that goes at least some way towards overcoming the above
disadvantages, or at least to provide the public with a useful
choice.
SUMMARY OF THE INVENTION
[0016] In a particular aspect of the invention, there is provided a
method of sustaining viability of a microbial culture of
carboxydotrophic bacteria, wherein a substrate comprising CO is
limited or unavailable, the method comprising the step of
maintaining the culture at a temperature or within a temperature
range below the optimum operating temperature for growth and/or
product production of the microbial culture.
[0017] In particular embodiments, the microbial culture is
suspended in a liquid nutrient medium.
[0018] A substrate is considered to be limited when there is
insufficient CO available to sustain growth and/or metabolite
production by the microbial culture. For example in a continuous
culture, the substrate is considered to be limited when steady
state growth cannot be sustained.
[0019] Typically, the substrate comprising CO is consumed by a
microbial culture at a rate of at least at least 0.1 mmol/g
microbial cells/minute; or at least 0.2 mmol/g/minute; or at least
0.3 mmol/g/minute; or at least 0.4 mmol/g/minute; or at least 0.5
mmol/g/minute. As such, in particular embodiments, the substrate
comprising CO is limited if less than at least 0.1 mmol/g microbial
cells/minute; or at least 0.2 mmol/g/minute; or at least 0.3
mmol/g/minute; or at least 0.4 mmol/g/minute; or at least 0.5
mmol/g/minute is available to the microbial culture. Limitation of
the substrate is typically associated with a cessation or slowing
of growth of the micro-organism.
[0020] In certain embodiments of the invention, the temperature of
the microbial culture is reduced to at least 5.degree.; or at least
10.degree.; or at least 15.degree.; or at least 20.degree.; or at
least 25.degree.; or at least 30.degree. below the optimum
operating temperature of the microbial culture. Those skilled in
the art will appreciate upon consideration of the instant
disclosure the optimum operating temperature of a carboxydotrophic
bacteria. However, by way of example, Clostridium autoethanogenum
has an optimum operating temperature of 37.degree. C. As such, in
particular embodiments of the invention, the temperature of the
microbial culture is reduced to less than 32.degree. C., or less
than 30.degree. C., or less than 25.degree. C., or less than
20.degree. C., or less than 15.degree. C., or less than 10.degree.
C., or less than 5.degree. C.
[0021] In particular embodiments of the invention, the temperature
of the microbial culture can be reduced by cooling the liquid
nutrient medium directly or indirectly. In particular embodiments,
at least a portion of the liquid nutrient medium may be passed
through a heat exchanging means to cool the liquid. Additionally,
or alternatively, the microbial culture is contained within a
vessel such as a bioreactor or a transport vessel, and the vessel
can be cooled by any known cooling means, such as a cooling
jacket.
[0022] In particular embodiments, the viability of the microbial
culture can be substantially maintained at reduced temperature for
at least 3 h, or at least 5 h, or at least 7 h, or at least 15 h,
or at least 30 h, or at least 48 h.
[0023] In another aspect of the invention, there is provided a
method of storing a microbial culture of a carboxydotrophic
bacteria, wherein a substrate stream is limited or unavailable, the
method comprising the step of reducing the temperature of the
microbial culture below the optimum operating temperature.
[0024] In particular embodiments, following storage, for example
when a substrate stream comprising sufficient CO is restored, the
temperature of the microbial culture is increased to the optimum
operating temperature. In such embodiments, the viability of the
microbial culture is substantially sustained throughout cooling,
storage and warming.
[0025] In another aspect, there is provided a method of sustaining
viability of a microbial culture during storage, the method
including the steps of: [0026] cooling the microbial culture to a
temperature or temperature range below the optimum operating
temperature, [0027] storing the microbial culture for a period of
time, In particular embodiments, the method includes warming the
culture to the optimum operating temperature following storage.
[0028] In particular embodiments, the extended period is at least 3
h, or at least 5 h, or at least 7 h, or at least 15 h, or at least
30 h, or at least 48 h.
[0029] In particular embodiments, the method is used to sustain
viability of a culture during periods of limited CO supply. In
another embodiment, the method can be used to sustain viability of
a microbial culture during transport to a remote location. In such
embodiments, it is considered there may be an insufficient CO
supply and/or inadequate agitation to sustain viability. As such,
cooling the microbial culture sustains viability for an extended
period.
[0030] In particular embodiments of the preceding aspects, storage
of the culture includes embodiments wherein the culture is
maintained in a bioreactor under limited substrate conditions.
Additionally or alternatively, the culture can be transferred from
a bioreactor to a storage vessel and/or transport vessel. It is
expected that in such embodiments, the culture can be returned to a
bioreactor at a later time.
[0031] In particular embodiments, the microbial culture can be used
to inoculate a bioreactor following storage. In such embodiments,
the microbial culture may be warmed to the optimum operating
temperature prior to, during or after inoculation.
[0032] In another aspect of the invention, there is provided a
method of transporting an inoculum comprising a microbial culture
of carboxydotrophic bacteria, the method including: [0033] cooling
the microbial culture to below the optimum operating temperature,
[0034] transporting the microbial culture to a remote location,
[0035] inoculating a bioreactor with the microbial culture.
[0036] In particular embodiments, the microbial culture is
transported to a remote location in a transport vessel. In
particular embodiments, the microbial culture can be cooled and/or
warmed in the transport vessel.
[0037] In particular embodiments, the method included pressurising
the transport vessel with a substrate comprising CO. In particular
embodiments, the transport vessel includes mixing means.
[0038] Embodiments of the invention find particular application in
the production of acids and alcohols, such as ethanol by
fermentation of a gaseous substrate comprising CO. The substrate
may comprise a gas obtained as a by-product of an industrial
process. In certain embodiments, the industrial process is selected
from the group consisting of ferrous metal products manufacturing,
non-ferrous products manufacturing, petroleum refining processes,
gasification of biomass, gasification of coal, electric power
production, carbon black production, ammonia production, methanol
production and coke manufacturing. In one embodiment of the
invention, the gaseous substrate is syngas. In one embodiment, the
gaseous substrate comprises a gas obtained from a steel mill.
[0039] The CO-containing substrate will typically contain a major
proportion of CO, such as at least about 20% to about 100% CO by
volume, from 40% to 95% CO by volume, from 40% to 60% CO by volume,
and from 45% to 55% CO by volume. In particular embodiments, the
substrate comprises about 25%, or about 30%, or about 35%, or about
40%, or about 45%, or about 50% CO, or about 55% CO, or about 60%
CO by volume. Substrates having lower concentrations of CO, such as
6%, may also be appropriate, particularly when H.sub.2 and CO.sub.2
are also present.
[0040] In various embodiments, the fermentation is carried out
using a culture of one or more strains of carboxydotrophic
bacteria. In various embodiments, the carboxydotrophic bacterium is
selected from Clostridium, Moorella, Oxobacter, Peptostreptococcus,
Acetobacterium, Eubacterium or Butyribacterium. In one embodiment,
the carboxydotrophic bacterium is Clostridium autoethanogenum.
[0041] The methods of the invention can be used to produce any of a
variety of alcohols, including without limitation ethanol and/or
butanol, by anaerobic fermentation of acids in the presence of
substrates, particularly gaseous substrates containing carbon
monoxide. The methods of the invention can also be applied to
aerobic fermentations, to anaerobic or aerobic fermentations of
other products, including but not limited to isopropanol, and to
fermentation of substrates other than carbon containing gases.
[0042] In another aspect, there is provided a system for
fermentation of a substrate comprising CO, including at least one
bioreactor; determining means adapted to determine whether the
substrate comprising CO is provided to a microbial culture is
limited or non-limited; and temperature control means configured
such that, in use, the temperature of the bioreactor can be
adjusted in response to determination of whether the supply of the
substrate comprising CO to the microbial culture is limited or
non-limited.
[0043] In particular embodiments, the controlling means are
configured to reduce the temperature of the bioreactor if the
determining means determines the supply of the substrate comprising
CO is limited. In particular embodiments, the system includes
processing means configured such that the temperature of the
bioreactor can be automatically regulated in response to changes in
whether the substrate comprising CO is limited or non-limited.
[0044] In another embodiment, the temperature control mean is
configured such that the temperature of the bioreactor can be
maintained at or about the optimum operating temperature if the
substrate is not limiting.
[0045] The invention may also includes the parts, elements and
features referred to or indicated in the specification of the
application, individually or collectively, in any or all
combinations of two or more of said parts, elements or features,
and where specific integers are mentioned herein which have known
equivalents in the art to which the invention relates, such known
equivalents are deemed to be incorporated herein as if individually
set forth.
DETAILED DESCRIPTION OF THE INVENTION
[0046] In accordance with the methods of the invention, it has been
surprisingly recognised that carboxydotrophic microbial cultures
may be stored with minimal or no additional substrate feeding
and/or agitation, at temperatures below their optimum. Accordingly,
in particular embodiments, the microbial culture can be transported
from one location to a remote location at temperatures
substantially below their growth and/or metabolite production
optimum temperature, and may be subsequently used to inoculate a
bioreactor. Typically, when a carboxydotrophic microbial culture is
stored without providing additional substrate and/or agitation, the
microbial culture will rapidly deplete any CO dissolved in a liquid
nutrient medium and viability of the culture deteriorates over
time. Consequently, when such cultures are used to inoculate a
bioreactor following storage without agitation, there may be a lag
time before microbial growth and/or expected productivity is
observed and/or the inoculation may be unsuccessful.
[0047] Additionally or alternatively, in particular embodiments,
where a substrate comprising CO, for example a gaseous substrate
stream, is not continuously available, the microbial culture can be
cooled to a temperature below the optimum operating temperature and
stored until further substrate is available. In other embodiments
where the substrate is limited (i.e. where CO is available but not
enough to promote optimum growth and/or metabolite production), the
microbial culture may be cooled to reduce the requirement for
CO.
[0048] In accordance with particular embodiments of the invention,
the method can be used to sustain viability of a microbial culture
through periods of limited substrate supply. For example,
continuous steady state fermentation of a substrate comprising CO
typically requires the substrate to be provided in a non-limited
manner such that a substantially constant growth and metabolite
production rate is sustained. However, the methods of the invention
can be used to sustain the viability of the culture during periods
of limited substrate supply which would otherwise result in culture
deterioration.
[0049] Without wishing to be bound by theory, in order to sustain
viability of carboxydotrophic bacteria such as Clostridium
autoethanogenum, CO needs to be supplied to the culture at a rate
greater than or equal to the CO uptake rate of the microbial
culture. For example, under optimum conditions required to promote
growth and/or metabolite production, the CO uptake rate of the
microbial culture is at least 0.1 mmol/g microbial cells/minute; or
at least 0.2 mmol/g/minute; or at least 0.3 mmol/g/minute; or at
least 0.4 mmol/g/minute; or at least 0.5 mmol/g/minute.
Accordingly, in particular embodiments where a microbial culture is
suspended in a liquid nutrient medium, the culture will rapidly
deplete CO dissolved in the medium unless the dissolved CO can be
replenished at a rate equal to or faster than the CO uptake rate.
Since CO is poorly soluble in aqueous nutrient media, an external
force, such as agitation and/or elevated pressure, is typically
required in addition to a constant supply of a substrate comprising
CO to maintain desirable CO transfer rate into solution. In
bioreactors, this is typically achieved by sparging CO into the
liquid nutrient medium and optionally further agitating the liquid
to increase the rate of CO transfer into the liquid. Such methods
are not generally available in vessels suitable for storage of a
microbial culture in a liquid nutrient medium, such as a transport
vessel.
[0050] It is recognised that in particular embodiments, wherein the
microbial culture is being transported from one location to a
remote location, there may be a small degree of agitation through
movement of the vessel. However, it is considered that the minor
agitation associated with vessel transport (i.e. transport by road)
is substantially less than what is required to maintain a CO
transfer rate into the liquid nutrient medium to prevent culture
deterioration.
[0051] In accordance with the methods of the invention, cooling the
microbial culture substantially sustains the viability of the
culture over an extended period. In particular embodiments, the
depletion of CO in a storage vessel can be minimised by cooling the
vessel. Additionally or alternatively, the microbial culture may be
allowed to cool towards a lower ambient temperature. Accordingly,
when such cultures are optionally returned to an optimum
temperature (or an optimum temperature range) and used to inoculate
a bioreactor following storage, microbial growth and/or desired
productivity is observed more quickly. Such methods ameliorate or
at least reduce the need for additional CO, sparging and/or
agitation.
DEFINITIONS
[0052] Unless otherwise defined, the following terms as used
throughout this specification are defined as follows:
[0053] The term "substrate comprising carbon monoxide" and like
terms should be understood to include any substrate in which carbon
monoxide is available to one or more strains of bacteria for growth
and/or fermentation, for example.
[0054] "Gaseous substrate 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.
[0055] In the context of fermentation products, the term "acid" as
used herein includes both carboxylic acids and the associated
carboxylate anion, such as the mixture of free acetic acid and
acetate present in a fermentation broth as described herein. The
ratio of molecular acid to carboxylate in the fermentation broth is
dependent upon the pH of the system. The term "acetate" includes
both acetate salt alone and a mixture of molecular or free acetic
acid and acetate salt, such as the mixture of acetate salt and free
acetic acid present in a fermentation broth as may be described
herein. The ratio of molecular acetic acid to acetate in the
fermentation broth is dependent upon the pH of the system.
[0056] 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 Reactor (ICR), Trickle Bed Reactor (TBR),
Bubble Column, Gas Lift Fermenter, Membrane Reactor such as Hollow
Fibre Membrane Bioreactor (HFMBR), Static Mixer, or other vessel or
other device suitable for gas-liquid contact.
[0057] 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 process. As will be
described further herein, in some embodiments the bioreactor may
comprise a first growth reactor and a second fermentation reactor.
As such, the addition of metals or compositions to a fermentation
reaction should be understood to include addition to either or both
of these reactors.
[0058] Unless the context requires otherwise, the phrases "storage"
and "store" are used in reference to periods when a microbial
culture has a limited substrate supply or a substrate is
unavailable. As such, the term includes periods when a microbial
culture under steady state growth conditions is temporarily
unavailable limited in substrate supply and includes periods when a
microbial culture is transferred from a bioreactor into a storage
vessel, such as an inoculum transfer vessel.
[0059] The term "overall net conversion" and the like, as used
herein, is intended to describe the conversion of substrates, such
as CO, to products including acid(s) and/or alcohol(s) by a
microbial culture at a particular time point. It is recognised that
portions of a microbial culture may be devoted to different
functions at a particular time point and a number of products may
be produced. Furthermore, one or more of the products present in
the fermentation broth may be converted into other products.
Accordingly, the overall net conversion includes all the products
produced by the microbial culture at any particular point in
time.
[0060] While the following description focuses on particular
embodiments of the invention, namely the production of ethanol
and/or acetate using CO as the primary substrate, it should be
appreciated that the invention may be applicable to production of
alternative alcohols and/or acids and the use of alternative
substrates as will be known by persons of ordinary skill in the art
to which the invention relates. For example, gaseous substrates
containing carbon dioxide and hydrogen may be used. Further, the
invention may be applicable to fermentation to produce butyrate,
propionate, caproate, ethanol, propanol, and butanol. The methods
may also be of use in producing hydrogen. By way of example, these
products may be produced by fermentation using microbes from the
genus Moorella, Clostridia, Ruminococcus, Acetobacterium,
Eubacterium, Butyribacterium, Oxobacter, Methanosarcina, and
Desulfotomaculum.
[0061] Certain embodiments of the invention are adapted to use gas
streams produced by one or more industrial processes. Such
processes include steel making processes, particularly processes
which produce a gas stream having a high CO content or a CO content
above a predetermined level (i.e., 5%). According to such
embodiments, acetogenic bacteria are preferably used to produce
acids and/or alcohols, particularly ethanol or butanol, within one
or more bioreactors. Those skilled in the art will be aware upon
consideration of the instant disclosure that the invention may be
applied to various industries or waste gas streams, including those
of vehicles with an internal combustion engine. Also, those skilled
in the art will be aware upon consideration of the instant
disclosure that the invention may be applied to other fermentation
reactions including those using the same or different
micro-organisms. It is therefore intended that the scope of the
invention is not limited to the particular embodiments and/or
applications described but is instead to be understood in a broader
sense; for example, the source of the gas stream is not limiting,
other than that at least a component thereof is usable to feed a
fermentation reaction. The invention has particular applicability
to improving the overall carbon capture and/or production of
ethanol and other alcohols from gaseous substrates such as
automobile exhaust gases and high volume CO-containing industrial
flue gases.
Fermentation
[0062] Processes for the production of ethanol and other alcohols
from gaseous substrates are known. Exemplary processes include
those described for example in WO2007/117157, WO2008/115080, U.S.
Pat. No. 6,340,581, U.S. Pat. No. 6,136,577, U.S. Pat. No.
5,593,886, U.S. Pat. No. 5,807,722 and U.S. Pat. No. 5,821,111,
each of which is incorporated herein by reference.
[0063] A number of anaerobic bacteria are known to be capable of
carrying out the fermentation of CO to alcohols, including
n-butanol and ethanol, and acetic acid, and are suitable for use in
the process of the present invention. Examples of such bacteria
that are suitable for use in the invention include those of the
genus Clostridium, such as strains of Clostridium Ijungdahlii,
including those described in WO 00/68407, EP 117309, U.S. Pat. Nos.
5,173,429, 5,593,886, and 6,368,819, WO 98/00558 and WO 02/08438,
Clostridium carboxydivorans (Liou et al., International Journal of
Systematic and Evolutionary Microbiology 33: pp 2085-2091),
Clostridium ragsdalei (WO/2008/028055) and Clostridium
autoethanogenum (Abrini et al, Archives of Microbiology 161: pp
345-351). Other suitable bacteria include those of the genus
Moorella, including Moorella sp HUC22-1, (Sakai et al,
Biotechnology Letters 29: pp 1607-1612), and those of the genus
Carboxydothermus (Svetlichny, V. A., Sokolova, T. G. et al (1991),
Systematic and Applied Microbiology 14: 254-260). Further examples
include Moorella thermoacetica, Moorella thermoautotrophica,
Ruminococcus productus, Acetobacterium woodii, Eubacterium limosum,
Butyribacterium methylotrophcum, Oxobacter pfennigii,
Methanosarcina barkeri, Methanosarcina acetivorans,
Desulfotomaculum kuznetsovii (Simpa et. al. Critical Reviews in
Biotechnology, 2006 Vol. 26. Pp 41-65). In addition, it should be
understood that other acetogenic anaerobic bacteria may be
applicable to the present invention as would be understood by a
person of skill in the art. It will also be appreciated that the
invention may be applied to a mixed culture of two or more
bacteria.
[0064] One exemplary micro-organism suitable for use in the present
invention is Clostridium autoethanogenum. In one embodiment, the
Clostridium autoethanogenum is a Clostridium autoethanogenum having
the identifying characteristics of the strain deposited at the
German Resource Centre for Biological Material (DSMZ) under the
identifying deposit number 19630. In another embodiment, the
Clostridium autoethanogenum is a Clostridium autoethanogenum having
the identifying characteristics of DSMZ deposit number DSMZ
10061.
[0065] Culturing of the bacteria used in the methods of the
invention may be conducted using any number of processes known in
the art for culturing and fermenting substrates using anaerobic
bacteria. Exemplary techniques are provided in the "Examples"
section below. By way of further example, those processes generally
described in the following articles using gaseous substrates for
fermentation may be utilised: (i) K. T. Klasson, et al. (1991).
Bioreactors for synthesis gas fermentations resources. Conservation
and Recycling, 5; 145-165; (ii) K. T. Klasson, et al. (1991).
Bioreactor design for synthesis gas fermentations. Fuel. 70.
605-614; (iii) K. T. Klasson, et al. (1992). Bioconversion of
synthesis gas into liquid or gaseous fuels. Enzyme and Microbial
Technology. 14; 602-608; (iv) J. L. Vega, et al. (1989). Study of
Gaseous Substrate Fermentation Carbon Monoxide Conversion to
Acetate. 2. Continuous Culture. Biotech. Bioeng. 34. 6. 785-793;
(v) J. L. Vega, et al. (1989). Study of gaseous substrate
fermentations: Carbon monoxide conversion to acetate. 1. Batch
culture. Biotechnology and Bioengineering. 34. 6. 774-784; (vi) J.
L. Vega, et al. (1990). Design of Bioreactors for Coal Synthesis
Gas Fermentations. Resources, Conservation and Recycling. 3.
149-160; all of which are incorporated herein by reference.
[0066] The fermentation may be carried out in any suitable
bioreactor, such as a continuous stirred tank reactor (CSTR), an
immobilised cell reactor, a gas-lift reactor, a bubble column
reactor (BCR), a membrane reactor, such as a Hollow Fibre Membrane
Bioreactor (HFMBR) or a trickle bed reactor (TBR). Also, in some
embodiments of the invention, the bioreactor may comprise a first,
growth reactor in which the micro-organisms are cultured, and a
second, fermentation reactor, to which fermentation broth from the
growth reactor is fed and in which most of the fermentation product
(e.g. ethanol and acetate) is produced.
[0067] According to various embodiments of the invention, the
carbon source for the fermentation reaction is a gaseous substrate
containing CO. The substrate may be a CO-containing waste gas
obtained as a by-product of an industrial process, or from some
another source such as from automobile exhaust fumes. In certain
embodiments, the industrial process is selected from the group
consisting of ferrous metal products manufacturing, such as a steel
mill, non-ferrous products manufacturing, petroleum refining
processes, gasification of coal, electric power production, carbon
black production, ammonia production, methanol production and coke
manufacturing. In these embodiments, the CO-containing substrate
may be captured from the industrial process before it is emitted
into the atmosphere, using any convenient method. Depending on the
composition of the CO-containing substrate, it may also be
desirable to treat it to remove any undesired impurities, such as
dust particles before introducing it to the fermentation. For
example, the gaseous substrate may be filtered or scrubbed using
known methods.
[0068] Alternatively, the CO-containing substrate may be sourced
from the gasification of biomass. The process of gasification
involves partial combustion of biomass in a restricted supply of
air or oxygen. The resultant gas typically comprises mainly CO and
H.sub.2, with minimal volumes of CO.sub.2, methane, ethylene and
ethane. For example, biomass by-products obtained during the
extraction and processing of foodstuffs such as sugar from
sugarcane, or starch from maize or grains, or non-food biomass
waste generated by the forestry industry may be gasified to produce
a CO-containing gas suitable for use in the present invention.
[0069] The CO-containing substrate will typically contain a major
proportion of CO, such as at least about 20% to about 100% CO by
volume, from 40% to 95% CO by volume, from 60% to 90% CO by volume,
and from 70% to 90% CO by volume. In particular embodiments, the
substrate comprises 25%, or 30%, or 35%, or 40%, or 45%, or 50% CO
by volume. Substrates having lower concentrations of CO, such as
6%, may also be appropriate, particularly when H.sub.2 and CO.sub.2
are also present.
[0070] 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 H2:CO. In other embodiments, the substrate
stream comprises low concentrations of H2, 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.
[0071] Typically, the carbon monoxide will be added to the
fermentation reaction in a gaseous state. However, the methods of
the invention are not limited to addition of the substrate in this
state. For example, the carbon monoxide can be provided in a
liquid. For example, a liquid may be saturated with a carbon
monoxide containing gas and that liquid added to the bioreactor.
This may be achieved using standard methodology. By way of example
a microbubble dispersion generator (Hensirisak et. al. Scale-up of
microbubble dispersion generator for aerobic fermentation; Applied
Biochemistry and Biotechnology Volume 101, Number 3/October, 2002)
could be used for this purpose.
[0072] It will be appreciated that for growth of the bacteria and
CO-to-alcohol fermentation to occur, in addition to the
CO-containing substrate gas, a suitable liquid nutrient medium will
need to be fed to the bioreactor. A nutrient medium will contain
vitamins and minerals sufficient to permit growth of the
micro-organism used. Anaerobic media suitable for the fermentation
of ethanol using CO as the sole carbon source are known in the art.
For example, suitable media are described in U.S. Pat. Nos.
5,173,429 and 5,593,886 and WO 02/08438, WO2007/115157 and
WO2008/115080 referred to above. The present invention provides a
novel media which has increased efficacy in supporting growth of
the micro-organisms and/or alcohol production in the fermentation
process. This media will be described in more detail
hereinafter.
[0073] The fermentation should desirably be carried out under
appropriate conditions for the desired fermentation to occur (e.g.
CO-to-ethanol). Reaction conditions that should be considered
include 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 CO in the liquid phase does
not become limiting, and maximum product concentrations to avoid
product inhibition. Suitable conditions are described in
WO02/08438, WO07/117,157 and WO08/115,080.
[0074] The optimum reaction conditions will depend partly on the
particular micro-organism used. However, in general, it is
preferred that the fermentation be performed at pressure higher
than ambient pressure. Operating at increased pressures allows a
significant increase in the rate of CO transfer from the gas phase
to the liquid phase where it can be taken up by the micro-organism
as a carbon source for the production of ethanol. 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.
[0075] Also, since a given CO-to-ethanol conversion rate is in part
a function of the 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. According to examples given in
U.S. Pat. No. 5,593,886, reactor volume can be reduced in linear
proportion to increases in reactor operating pressure, i.e.
bioreactors operated at 10 atmospheres of pressure need only be one
tenth the volume of those operated at 1 atmosphere of pressure.
[0076] The benefits of conducting a gas-to-ethanol fermentation at
elevated pressures have also been described elsewhere. For example,
WO 02/08438 describes gas-to-ethanol fermentations performed under
pressures of 30 psig and 75 psig, giving ethanol productivities of
150 g/l/day and 369 g/l/day respectively. However, example
fermentations performed using similar media and input gas
compositions at atmospheric pressure were found to produce between
10 and 20 times less ethanol per litre per day.
[0077] It is also desirable that the rate of introduction of the
CO-containing gaseous substrate is such as to ensure that the
concentration of CO in the liquid phase does not become limiting.
This is because a consequence of CO-limited conditions may be that
the ethanol product is consumed by the culture.
Product Recovery
[0078] The products of the fermentation reaction can be recovered
using known methods. Exemplary methods include those described in
WO07/117,157, WO08/115,080, U.S. Pat. No. 6,340,581, U.S. Pat. No.
6,136,577, U.S. Pat. No. 5,593,886, U.S. Pat. No. 5,807,722 and
U.S. Pat. No. 5,821,111. However, briefly and by way of example
only ethanol may be recovered from the fermentation broth by
methods such as fractional distillation or evaporation, and
extractive fermentation.
[0079] Distillation of ethanol from a fermentation broth yields an
azeotropic mixture of ethanol and water (i.e., 95% ethanol and 5%
water). Anhydrous ethanol can subsequently be obtained through the
use of molecular sieve ethanol dehydration technology, which is
also well known in the art.
[0080] Extractive fermentation procedures involve the use of a
water-miscible solvent that presents a low toxicity risk to the
fermentation organism, to recover the ethanol from the dilute
fermentation broth. For example, oleyl alcohol is a solvent that
may be used in this type of extraction process. Oleyl alcohol is
continuously introduced into a fermenter, whereupon this solvent
rises forming a layer at the top of the fermenter which is
continuously extracted and fed through a centrifuge. Water and
cells are then readily separated from the oleyl alcohol and
returned to the fermenter while the ethanol-laden solvent is fed
into a flash vaporization unit. Most of the ethanol is vaporized
and condensed while the oleyl alcohol is non volatile and is
recovered for re-use in the fermentation.
[0081] Acetate, which is produced as a by-product in the
fermentation reaction, may also be recovered from the fermentation
broth using methods known in the art.
[0082] For example, an adsorption system involving an activated
charcoal filter may be used. In this case, it is preferred that
microbial cells are first removed from the fermentation broth using
a suitable separation unit. Numerous filtration-based methods of
generating a cell free fermentation broth for product recovery are
known in the art. The cell free ethanol--and acetate--containing
permeate is then passed through a column containing activated
charcoal to adsorb the acetate. Acetate in the add form (acetic
acid) rather than the salt (acetate) form is more readily adsorbed
by activated charcoal. It is therefore preferred that the pH of the
fermentation broth is reduced to less than about 3 before it is
passed through the activated charcoal column, to convert the
majority of the acetate to the acetic acid form.
[0083] Acetic acid adsorbed to the activated charcoal may be
recovered by elution using methods known in the art. For example,
ethanol may be used to elute the bound acetate. In certain
embodiments, ethanol produced by the fermentation process itself
may be used to elute the acetate. Because the boiling point of
ethanol is 78.8.degree. C. and that of acetic acid is 107.degree.
C., ethanol and acetate can readily be separated from each other
using a volatility-based method such as distillation.
[0084] Other methods for recovering acetate from a fermentation
broth are also known in the art and may be used in the processes of
the present invention. For example, U.S. Pat. Nos. 6,368,819 and
6,753,170 describe a solvent and cosolvent system that can be used
for extraction of acetic acid from fermentation broths. As with the
example of the oleyl alcohol-based system described for the
extractive fermentation of ethanol, the systems described in U.S.
Pat. Nos. 6,368,819 and 6,753,170 describe a water immiscible
solvent/co-solvent that can be mixed with the fermentation broth in
either the presence or absence of the fermented micro-organisms in
order to extract the acetic acid product. The solvent/co-solvent
containing the acetic acid product is then separated from the broth
by distillation. A second distillation step may then be used to
purify the acetic acid from the solvent/co-solvent system.
[0085] The products of the fermentation reaction (for example
ethanol and acetate) may be recovered from the fermentation broth
by continuously removing a portion of the broth from the
fermentation bioreactor, separating microbial cells from the broth
(conveniently by filtration), and recovering one or more product
from the broth simultaneously or sequentially. In the case of
ethanol it may be conveniently recovered by distillation, and
acetate may be recovered by adsorption on activated charcoal, using
the methods described above. The separated microbial cells are
preferably returned to the fermentation bioreactor. The cell free
permeate remaining after the ethanol and acetate have been removed
is also preferably returned to the fermentation bioreactor.
Additional nutrients (such as B vitamins) may be added to the cell
free permeate to replenish the nutrient medium before it is
returned to the bioreactor. Also, if the pH of the broth was
adjusted as described above to enhance adsorption of acetic acid to
the activated charcoal, the pH should be re-adjusted to a similar
pH to that of the broth in the fermentation bioreactor, before
being returned to the bioreactor.
Sustaining Culture Viability
[0086] In accordance with the invention, there is provided, a
method of substantially sustaining viability of a microbial culture
of carboxydotrophic bacteria, wherein substrate comprising CO is
limited or unavailable, the method comprising reducing the
temperature of the microbial culture below the optimum temperature
for growth and/or product production.
[0087] In accordance with the methods of the invention, culture
viability is sustained if, on warming to optimum temperature (or
range), the culture can resume metabolism to produce products
and/or cell growth. In particular embodiments, the microbial
culture can be used to inoculate a bioreactor and sustaining
culture viability ensures the culture can resume metabolism
following transfer. It is recognised that a microbial culture
stored in accordance with the methods of the invention may continue
to metabolise and/or grow albeit at a slower rate. However, on
restoring the temperature of the microbial culture toward the
optimum, metabolism and/or growth rate are expected to increase to
pre-storage levels.
[0088] A microbial culture becomes limited in CO when the rate of
transfer of CO into an aqueous nutrient medium is slower than the
rate at which the microbial culture can take up (or consume) the
CO. Typically, carboxydotrophic bacteria, such as Clostridium
autoethanogenum, uptake CO from a liquid nutrient medium at a rate
greater than 0.1 mmol/g microbial cells/minute; or at least 0.2
mmol/g/minute; or at least 0.3 mmol/g/minute; or at least 0.4
mmol/g/minute; or at least 0.5 mmol/g/minute. Accordingly, it is
generally necessary to provide a constant stream of a substrate
comprising CO to the microbial culture. Furthermore, due to the low
solubility of CO in aqueous systems, it is typically necessary to
further increase the CO transfer rates (mass transfer), for example
by increasing partial pressure of CO in the substrate stream and/or
agitation of the liquid nutrient medium. Upon consideration of the
instant disclosure, those skilled in the art will appreciate
alternative methods of increasing mass transfer of CO in accordance
with particular embodiments of the invention.
[0089] In particular embodiments, the methods of the invention can
be used to sustain the viability of a microbial culture, wherein
the microbial culture is limited in CO, such that the rate of
transfer of CO into solution is less than the uptake rate of the
culture. Such situations may arise when a substrate comprising CO
is not continuously provided to the microbial culture; the mass
transfer rate is low; or there is insufficient CO in a substrate
stream to sustain culture vitality at optimum temperature. In such
embodiments, the microbial culture will rapidly deplete the CO
dissolved in the liquid nutrient medium and become substrate
limited as further substrate cannot be provided fast enough. Unless
the microbial culture is cooled in accordance with the methods of
the invention, the viability of the microbial culture will diminish
over time, resulting in complete culture death, or the culture
deteriorating to such a level where it is no longer limited by the
conditions.
[0090] For example, in particular embodiments, a microbial culture
comprising one or more carboxydotrophic micro-organisms can be
operated under substantially steady state conditions when a
substrate comprising CO is not limited. Under such conditions, it
is expected the microbial culture will have a substantially
constant growth rate and substantially constant metabolite(s)
production rate. However, when the substrate cannot be provided in
a non-limited way, microbial growth will slow or cease and the
microbial culture will rapidly deteriorate and will wash out of a
continuously purged bioreactor. Under such conditions, even if the
substrate is returned to a non-limited supply, the culture may not
be revived, or at least revival takes an extended period. However,
in accordance with the invention, the temperature of the culture is
decreased, such that viability of the microbial culture is
sustained during storage periods of limited or no substrate
supply.
[0091] It is recognised that the metabolism of the microbial
culture may slow when the temperature is decreased, so operating
conditions, such as cell retention times may need to be
adjusted.
[0092] In particular embodiments of the invention, the stored
microbial culture is used for inoculation of a bioreactor. In such
embodiments, it is desirable that the culture is suitably dense
(i.e. large number of microbes per unit volume) and that the
viability of the culture is substantially sustained during storage
(i.e. transport to a remote location). Typically, the higher the
density of the microbial cells in the culture, the faster they will
deplete any CO available in a liquid nutrient medium. Without
wishing to be bound by theory, it is considered that when CO is not
available, or is sufficiently depleted, the viability of the
microbial culture decreases. For example, at least a portion of the
culture begins to die off and/or the culture switches to a slower
metabolism, such that when a bioreactor is inoculated with the
microbial culture, there is a lag before high growth rates and/or
productivity is attained. However, when the culture is cooled, the
depletion of CO in the liquid nutrient medium is slowed such that
the culture viability is substantially preserved over an extended
period.
[0093] In accordance with the methods of the invention, the culture
may be cooled to a temperature below the optimum growth and/or
metabolite production temperature, such that viability of the
culture is sustained over an extended period. Typically,
carboxydotrophic micro-organisms have an optimum operating
temperatures of carboxydotrophic bacteria in the range
30-70.degree. C. Examples of optimum operating temperature are
detailed in "Microbiology of synthesis gas fermentation for
biofuels production" A. M. Henstra et al. Current Opinion in
Biotechnology, 2007, 18, 200-206. For example, mesophilic bacteria,
such as Clostridium autoethanogenum, Clostridium Ijungdahli and
Clostridium carboxydivorans have an optimum growth and metabolite
production temperature of approximately 37.degree. C. However,
thermophilic bacteria have significantly higher optimum
temperatures of 55-70.degree. C., for example strains of Moorella
thermoacetica (55-60.degree. C.), Carboxydothermus hydrogenoformans
(70-72.degree. C.), Desulfotomaculum carboxydivorans (60.degree.
C.). As such, in accordance with the methods of the invention, it
is necessary to cool the microbial culture to at least 2.degree.;
or at least 5.degree.; or at least 10.degree.; at least 15.degree.;
or at least 20.degree.; or at least 25.degree.; or at least
30.degree. below the optimum temperature to sustain culture
viability. For example, Clostridium autoethanogenum can be cooled
to less than 30.degree. C., or less than 25.degree. C., or less
than 20.degree. C., or less than 15.degree. C., or less than
10.degree. C., or less than 5.degree. C.
[0094] In accordance with the methods of the invention, on cooling,
viability of the culture is sustained for extended periods, even in
the absence of additional substrate comprising CO and/or agitation.
In particular embodiments, viability of the culture is sustained
for at least 3 h, or at least 5 h, or at least 7 h, or at least 15
h, or at least 30 h, or at least 48 h. For example; Clostridium
autoethanogenum remains viable for at least 30 hours, when stored
at reduced temperature.
[0095] Those skilled in the art will appreciate means required to
cool a microbial culture will depend on several factors including
size and shape of the vessel containing the culture, speed at which
the culture is cooled and whether the culture is exothermic or
endothermic. For example, many large scale fermentation processes
need to be externally cooled to remove excess heat generated during
the fermentation reaction. The known cooling means already provided
may be adapted to further cool the microbial culture to sustain
viability. In alternative embodiments, where the microbial culture
requires external heating to maintain the optimum operating
temperature, the culture may be cooled by removing the heat source
and allowing the fermenter to cool to ambient temperature over
time. Additionally or alternatively, such cultures may be further
cooled using any known refrigeration or cooling means.
[0096] In particular embodiments of the invention, the liquid
nutrient media is allowed to cool below the optimum operating
temperature by removing thermostatic heat control. Under such
conditions, the temperature of the liquid nutrient media and the
microbial culture will fall toward ambient temperature over time.
In accordance with the invention, as the temperature of the
microbial culture falls below the optimum operating temperature,
alcohol productivity increases.
[0097] It is considered that periods where viability of a microbial
culture may be sustained using the methods of the invention will be
commonly encountered in industrial fermentation processes, as
continuity of a substrate stream comprising CO may not be
guaranteed. For example, where a substrate comprising CO is derived
from an industrial process, such as off-gas from a steel mill,
there may be occasions where the industrial process (i.e. steel
manufacture) is slowed or shut down for extended periods. Under
such conditions, the production of a substrate comprising CO will
slow or stop altogether. Consequently, when CO supply is limited or
CO is unavailable to a bioreactor containing a carboxydotrophic
microbial culture, the viability of the culture will diminish over
time. However, in accordance with the methods of the invention, if
the culture is cooled, the viability can be sustained during CO
limited operation.
[0098] Similarly, when syngas produced from the gasification of
feedstock's such as biomass or municipal solid waste is used as the
substrate stream, there may be times when the CO content of the
stream decreases, or the gasifier has to be taken off-line, for
maintenance (for example). Again, under such conditions, viability
of a microbial culture requiring CO for metabolism will deteriorate
unless the culture can be cooled in accordance with the methods of
the invention.
[0099] In an alternative embodiment, the methods of the invention
can be used to substantially sustain the viability of a microbial
culture used for inoculation of a remote bioreactor. For example, a
microbial culture can be placed in a vessel suitable for transport
and transported to a remote location. Typically, the transport
vessel would require a supply of CO and agitation means to ensure
viability of the culture was sustained, both of which can be
difficult to provide in mobile environments. However, in accordance
with the methods of the invention, the microbial culture can be
cooled in the transport vessel such that viability of the inoculum
is sustained during transport, even in the absence of a sufficient
supply of CO and/or agitation.
[0100] In accordance with another embodiment of the invention,
there is provided a system for fermentation of a substrate
comprising CO, including at least one bioreactor; determining means
adapted to determine whether the substrate comprising CO is
provided to a microbial culture is limited or non-limited; and
temperature control means configured such that, in use, the
temperature of the bioreactor can be adjusted in response to
determination of whether the supply of the substrate comprising CO
to the microbial culture is limited or non-limited.
[0101] In particular embodiments, wherein the determining means
determine that the substrate supply has become limited, the
temperature of the microbial culture can be decreased to sustain
culture viability. Additionally or alternatively, wherein the
determining means determines the substrate is not limited, the
temperature can be maintained substantially at optimum operating
temperature. In particular embodiments, the system includes
processing means, such that in use, the controlling means can
regulate the temperature of the microbial culture automatically in
accordance with the methods of the invention.
[0102] FIG. 1 is a schematic representation of a system 100
according to one embodiments of the invention. Input substrate
stream 1 enters bioreactor 2 via a suitable conduit. Input
substrate stream 1 comprises CO and in accordance with the methods
of the invention, the rate of supply and/or the composition of the
substrate stream 1 may vary. The system 100 includes determining
means 3 which, in use, determine whether the substrate supplied to
a microbial culture in the bioreactor is limited. The system 100
includes temperature control means 4, which can regulate the
temperature of the bioreactor 1 such that a microbial culture can
be maintained at an optimum operating temperature, or the
temperature decreased and/or maintained at a temperature below the
optimum operating temperature.
[0103] In particular embodiments, the temperature control means 4
is configured such that in use, if the determining means determines
the substrate supply is not limited, the temperature of the
fermentation can be maintained at or around the optimum operating
temperature. Additionally or alternatively, if the determining
means 3 determines that the substrate supply is limited, the
controlling means 4 can decrease the temperature of the bioreactor
1 in accordance with the methods of the invention. Thus, the
temperature can be controlled at a temperature substantially below
the optimum operating temperature until the substrate supply is no
longer limiting.
[0104] In particular embodiments, the system 100 includes optional
processing means 5 configured to regulate the controlling means 4
automatically, in response to determinations made by the
determining means 3.
EXAMPLES
Materials and Methods
Preparation of Media LM33:
TABLE-US-00001 [0105] Concentration per Media Component 1.0 L of
Media MgCl.sub.2.cndot.6H.sub.2O 0.5 g NaCl 0.2 g
CaCl.sub.2.cndot.6H.sub.2O 0.26 g NaH.sub.2PO.sub.4 2.04 g KCl 0.15
g NH.sub.4Cl 2.5 g Composite trace metal solution (LS06) 10 mL
Composite B vitamin solution (LS03) 10 mL Resazurin (2 g/L stock) 1
mL FeCl.sub.3 (5 g/L stock) 2 mL Cysteine HCl 0.5 g Distilled water
Up to 1 L
TABLE-US-00002 Composite B vitamin Solution (LS03) per L of Stock
Biotin 20.0 mg Folic acid 20.0 mg Pyridoxine hydrochloride 10.0 mg
Thiamine.cndot.HCl 50.0 mg Riboflavin 50.0 mg Nicotinic acid 50.0
mg Calcium D-(*)-pantothenate 50.0 mg Vitamin B12 50.0 mg
p-Aminobenzoic acid 50.0 mg Thioctic acid 50.0 mg Distilled water
To 1 Litre
TABLE-US-00003 Composite trace metal solution (LSO6) per L of stock
Nitrilotriacetic Acid 1.5 g MgSO.sub.4.cndot.7H.sub.2O 3.0 g
MnSO.sub.4.cndot.H.sub.2O 0.5 g NaCl 1.0 g
FeSO.sub.4.cndot.7H.sub.2O 0.1 g
Fe(SO.sub.4).sub.2(NH.sub.4).sub.2.cndot.6H.sub.2O 0.8 g
CoCl.sub.2.cndot.6H.sub.2O 0.2 g ZnSO.sub.4.cndot.7H.sub.2O 0.2 g
CuCl.sub.2.cndot.2H.sub.2O 0.02 g
AlK(SO.sub.4).sub.2.cndot.12H.sub.2O 0.02 g H.sub.3BO.sub.3 0.30 g
NaMoO.sub.4.cndot.2H.sub.2O 0.03 g Na.sub.2SeO.sub.3 0.02 g
NiCl.sub.2.cndot.6H.sub.2O 0.02 g Na.sub.2WO.sub.4.cndot.6H.sub.2O
0.02 g
[0106] Media was prepared at pH 5.5 as follows. All ingredients
with the exception of Cysteine-HCl were mixed in 400 ml distilled
water. This solution was made anaerobic by heating to boiling and
allowing it to cool to room temperature under a constant flow of N2
gas. Once cool, the Cysteine-HCl was added and the pH of the
solution adjusted to 5.5 before making the volume up to 1000 ml;
anaerobicity was maintained throughout the experiments.
[0107] Bacteria: Clostridium autoethanogenum were obtained from the
German Resource Centre for Biological Material (DSMZ). The
accession number given to the bacteria is DSMZ 19630.
Typical Continuous Culture in Bioreactor at Atmospheric Pressure
for Inoculum
[0108] A five-litre bioreactor was filled with 4900 ml of the media
LM33 without Composite B vitamin solution (LS03) or Cysteine-HCl
and autoclaved for 30 minutes at 121.degree. C. While cooling down,
the media was sparged with N2 to ensure anaerobicity. Cysteine-HCl
and Composite B vitamin solution (LS03) were then added.
Anaerobicity was maintained throughout the fermentation. The gas
was switched to 95% CO, 5% CO.sub.2 at atmospheric pressure prior
to inoculation with 100 ml of a Clostridium autoethanogenum
culture. The bioreactor was maintained at 37.degree. C. stirred at
200 rpm at the start of the culture. During the growth phase, the
agitation was increased to 400 rpm. The pH was set to 5.5 and
maintained by automatic addition of 5 M NaOH. Fresh anaerobic media
was continuously added into the bioreactor to maintain a defined
biomass and acetate level the bioreactor.
Example 1
[0109] Sterile serum bottles were purged three times with CO
containing gas (20% CO2; 30% N2 and 3% H2 in CO) and then evacuated
to a vacuum of -5 psi. 50 ml of active culture containing biomass,
acetate and traces of ethanol under atmospheric pressure was
transferred directly from a continuous bioreactor into the 234 ml
serum bottle. The 184 ml headspace was then filled with the CO
containing gas to 40 psia and incubated without shaking at the
indicated temperature.
[0110] After 3, 6, 24 and 31 hours of incubation, a 2 ml sample
from each serum vial was transferred into a new serum vial
containing 50 ml of media (LM33) prepared in accordance with the
above. The vials were filled with the CO containing gas to 40 psia
and incubated at 37.degree. C. for several days with constant
agitation.
[0111] Growth of the inoculated vials was visually assessed at time
intervals and -/+/++ were assigned to describe no growth, slight
growth and dense growth respectively (see Table 1).
TABLE-US-00004 TABLE 1 Growth of inoculated Clostridium
autoethanogenum culture following storage at various temperatures
over 3, 6, 24 and 31 h. Incubation time 3 h 6 h 24 h 31 h
Incubation Days following inoculation temp 0 1 2 0 1 2 0 1 2 0 1 2
4.degree. C. - + ++ - + ++ - + ++ - + ++ 14.degree. C. - + ++ - +
++ - + ++ - - + 24.degree. C. - + ++ - + + - - - - - - 37.degree.
C. - - - - - - - - - - - -
[0112] The optimum temperature for production of products and
microbial growth of Clostridium autoethanogenum is 37.degree. C. At
37.degree. C., the non shaken vials were either non-viable or had
substantially reduced viability when used for inoculation after 3,
6, 24 and 31 hours. It is considered that without agitation, the
active microbial culture rapidly depletes the limited CO dissolved
in the liquid nutrient medium. The excess carbon monoxide in the
headspace may have limited transfer into the liquid nutrient
medium. However, in the absence of agitation, it is expected there
will be a CO gradient, wherein the uppermost surface of the liquid
nutrient medium may have a relatively high CO concentration, but
this will decrease down through the medium. In the absence of
agitation, the microbial cells will settle to the bottom of the
vial, where they will be substantially starved of substrate and
will rapidly decrease in viability. Subsequently, the deteriorated
or dead culture is unsuitable for inoculation.
[0113] On reducing the temperature of the stored culture to
24.degree. C., the microbial culture remained substantially viable
for inoculation of a bioreactor for over 3 h. At 14.degree. C., the
microbial culture remained substantially viable following storage
for 3 h, 6 h and 24 h. Following 31 h storage, the microbial
culture remained viable, but took longer to grow following
inoculation. At 4.degree. C., the microbial culture remained viable
following storage over all times investigated.
[0114] The invention has been described herein with reference to
certain preferred embodiments, in order to enable the reader to
practice the invention without undue experimentation. Those skilled
in the art will appreciate that the invention is susceptible to
variations and modifications other than those specifically
described. It is to be understood that the invention includes all
such variations and modifications. Furthermore, titles, heading, or
the like are provided to enhance the reader's comprehension of this
document, and should not be read as limiting the scope of the
present invention. The entire disclosures of all applications,
patents and publications cited above and below, if any, are herein
incorporated by reference.
[0115] The reference to any prior art in this specification is not,
and should not be taken as, an acknowledgement or any form of
suggestion that that prior art forms part of the common general
knowledge in the field of endeavour in any country in the
world.
[0116] Throughout this specification and any claims which follow,
unless the context requires otherwise, the words "comprise",
"comprising" and the like, are to be construed in an inclusive
sense as opposed to an exclusive sense, that is to say, in the
sense of "including, but not limited to".
* * * * *