Note: Descriptions are shown in the official language in which they were submitted.
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METHOD FOR SUSTAINING MICROORGANISM CULTURE IN SYNGAS
FERMENTATION PROCESS IN DECREASED CONCENTRATION OR
ABSENCE OF VARIOUS SUBSTRATES
FIELD OF INVENTION
The present invention is directed to improvements in microbial fermentation
methods for the production of alcohol from a gaseous substrate containing at
least one
reducing gas containing at least one acetogenic microorganism.
BACKGROUND OF THE INVENTION
Numerous conventional methods exist for sustaining microorganism culture.
However, these methods suffer from numerous inefficiencies. There remains a
need for
additional more effective methods for sustaining microorganism cultures in the
absence
of various substrates in a syngas fermentation process.
Three strains of acetogens (Drake, 1994) have been described for use in the
production of liquid fuels from syngas: Butyribacterium methylotrophicum
(Grethlein et
al., 1990; Jain et al., 1994b); Clostridium autoethanogenum (Abrini et al.,
1994);
Clostridium ljungdahlii (Arora et al, 1995; Barik et al., 1988; Barik et al.
1990; and
Tanner et al., 1993). Of these, Clostridium ljungdahlii and Clostridium
autoethanogenum
are known to convert CO to ethanol.
U.S. Pat. No. 5,173,429 to Gaddy et al. discloses Clostridium ljungdahlii ATCC
No. 49587, an anaerobic microorganism that produces ethanol and acetate from
CO and
H2O and/or CO2 and H2 in synthesis gas.
U.S. Pat. No. 5,192,673 to Jain et al. discloses a mutant strain of
Clostridium
acetobytylicum and a process for making butanol with the strain.
U.S. Pat. No. 5,593,886 to Gaddy et al. discloses Clostridium ljungdahlii ATCC
No. 55380. This microorganism can anaerobically produce acetate and ethanol
using
waste gas (e.g. carbon black waste gas) as a substrate.
U.S. Pat. No. 5,807,722 to Gaddy et al. discloses a method and apparatus for
converting waste gases into useful products such as organic acids and alcohols
using
anaerobic bacteria, such as Clostridium ljungdahlii ATCC No. 55380.
U.S. Pat. No. 6,136,577 to Gaddy et al. discloses a method and apparatus for
converting waste gases into useful products such as organic acids and alcohols
(particularly ethanol) using anaerobic bacteria, such as Clostridium
ljungdahlii ATCC
Nos. 55988 and 55989.
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U.S. Pat. No. 6,136,577 to Gaddy et al. discloses a method and apparatus for
converting waste gases into useful products such as organic acids and alcohols
(particularly acetic acid) using anaerobic strains of Clostridium ljungdahlii.
U.S. Pat. No. 6,753,170 to Gaddy et al. discloses an anaerobic microbial
fermentation process for the production of acetic acid.
U.S. Pat. No. 7,285,402 to Gaddy et al. discloses an anaerobic microbial
fermentation process for the production of alcohol.
Other strains of aceotgens have also been described for use in the production
of
liquid fuels from synthesis gas, e.g.: Butyribacterium methylotrophicum
(Grethlein et al.,
1990, Appl. Biochem. Biotech. 24/24:875-884); and Clostridium autoethanogenum
(Abrini et al., 1994, Arch. Microbiol. 161:345-351).
There remains a need in the art in preserving culture in syngas fermenation
process in decreased concentration or absence of various substrates. There is
a need to
sustain cultures in the event of various interruptions in industrial process
of alcohol
production. Particularly, there remains a need to sustain microorganism
culture in the
event of decreased: CO, H2, or CO and H2 in various concentrations.
SUMMARY OF THE INVENTION
The present invention relates to methods for sustaining microorganism culture
in
a syngas fermentation reactor in decreased concentration or absence of various
substrates
comprising: adding carbon dioxide and optionally alcohol; maintaining free
acetic acid
concentrations; and performing the above mentioned steps within specified
time.
The present invention further contemplates a method for preventing rapid loss
of
microorganism culture in a syngas fermentation reactor in decreased
concentration or
absence of various substrates comprising: adding carbon dioxide and optionally
alcohol;
decreasing temperature from the operating temperature; maintaining free acetic
acid
concentrations; and performing the above mentioned steps within specified
time.
The present invention further provides a method for sustaining microorganism
culture in a syngas fermentation reactor due to decreased concentration or
absence of
various substrates in feed gas supply comprising: adding carbon dioxide and
optionally
alcohol; decreasing temperature from operating temperature; maintaining free
acetic acid
concentrations; and performing the above mentioned steps within specified
time.
As an embodiment of the present invention, alcohol can be utilized as a
substrate.
Although several alternative growth substrates were tried, none performed as
well as
alcohol and none are as readily available as the alcohol. When synthesis gas
supply is
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restored the microorganism culture readily returns to utilizing the syngas.
Additionally, as an embodiment, solely utilizing the acetate / alcohol pathway
does not
provide the opportunity for other competing bacteria to grow that may be
present in the
culture broth or process piping. Whereas a growth substrate such as glucose
would be
readily available to any organisms present for their growth.
Prior art would include adjustments to the culture broth to maintain a low
free
acetic acid concentration. These would include raising the pH and increasing
the liquid
flow to wash out the acetyl. As an embodiment, temperature reduction to reduce
culture
activity and using the product ethanol and carbon dioxide to provide energy
back to the
culture to maintain viability., Additionally, the concept of novel alternative
substrate.
This is an improvement on the process because there will be times when gas
supply is interrupted due to interruptions in gasifier feedstock supply,
conveying
equipment, drying equipment, gas cleanup or any other unit along the gas
supply line.
Another application of the present invention comprises transporting innoculum
from one
site to another. In transport the culture may not not have a syngas supply,
therefore an
alternative substrate would be required. Having the capability to maintain
viability for
12 hours or more would be an improvement in process capability. Therefore,
having an
alternative that is economically and technically viable will result in
minimizing
interruptions and/or downturns in alcohol production, plus plant startups and
restarts.
BRIEF DESCRIPTION OF THE FIGURE
FIG. 1 is a schematic diagram illustrating an embodiment of overall process
flow
contemplated during normal operations of the present invention. Although
ethanol is
indicated in the diagram, other alcohols are also contemplated by the present
invention.
FIG. 2 is a schematic diagram illustrating embodiments of the present
invention
showing trends with carbon dioxide addition, alcohol consumption, and culture
recovery.
FIG. 3 is a schematic diagram illustrating comparisons of the present
invention
demonstrating lack of alcohol consumption and lack of culture recovery.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
Unless otherwise defined, the following terms as used throughout this
specification are defined as follows.
The term "about" modifying any amount refers to the variation in that amount
encountered in real world conditions of sustaining microorganism culture,
e.g., in the lab,
pilot plant, or production facility. For example, an amount of an ingredient
employed in a
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mixture when modified by "about" includes the variation and degree of care
typically
employed in measuring in an experimental condition in production plant or lab.
For
example, the amount of a component of a product when modified by "about"
includes
the variation between batches in an multiple experiments in the plant or lab
and the
variation inherent in the analytical method. Whether or not modified by
"about," the
amounts include equivalents to those amounts. Any quantity stated herein and
modified
by "about" can also be employed in the present invention as the amount not
modified by
"about."
Unless stated otherwise, the term "acetate" is used to describe the mixture of
molecular or free acetic acid and acetate salt present in the fermentation
broth. The ratio
of molecular acetic acid to acetate is dependent upon the pH of the system,
i.e., at a
constant "acetate" concentration, the lower the pH, the higher the molecular
acetic acid
concentration relative to acetate salt.
The term "acetogen" or "acetogenic" refers to a bacterium that generates
acetate
as a product of anaerobic respiration. This process is different from acetate
fermentation,
although both occur in the absence of oxygen and produce acetate. These
organisms are
also referred to as acetogenic bacteria, because all known acetogens are
bacteria.
Acetogens are found in a variety of habitats, generally those that are
anaerobic (lack
oxygen). Acetogens can use a variety of compounds as sources of energy and
carbon; the
best studied form of acetogenic metabolism involves the use of carbon dioxide
as a
carbon source and hydrogen as an energy source.
The terms "bioreactor," "reactor," or "fermentation bioreactor," include a
fermentation device consisting of one or more vessels and/or towers or piping
arrangement, which includes the Continuous Stirred Tank Reactor (CSTR),
Immobilized
Cell Reactor (ICR), Trickle Bed Reactor (TBR), Bubble Column, Gas lift
Fermenter,
Static Mixer, or other device suitable for gas-liquid contact. Preferably for
the method of
this invention, the fermentation bioreactor comprises a growth reactor which
feeds the
fermentation broth to a second fermentation bioreactor, in which most of the
product,
ethanol, is produced.
"Cell concentration" in this specification is based on dry weight of bacteria
per
liter of sample. Cell concentration is measured directly or by calibration to
a correlation
with optical density.
The term "continuous method" as used herein refers to a fermentation method
which includes continuous nutrient feed, substrate feed, cell production in
the bioreactor,
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cell removal (or purge) from the bioreactor, and product removal. This
continuous feeds,
removals or cell production may occur in the same or in different streams. A
continuous
process results in the achievement of a steady state within the bioreactor. By
"steady
state" is meant that all of these measurable variables (i.e., feed rates,
substrate and
nutrient concentrations maintained in the bioreactor, cell concentration in
the bioreactor
and cell removal from the bioreactor, product removal from the bioreactor, as
well as
conditional variables such as temperatures and pressures) are constant over
time.
"Ethanol productivity" is the volumetric productivity of ethanol, calculated
as the
ratio of the steady state ethanol concentration and the liquid retention time
(LRT) in
continuous systems, or the ratio of the ethanol concentration and the time
required to
produce that concentration in batch systems. The phrase "high ethanol
productivity"
describes a volumetric ethanol productivity of greater than 10 g/Lday.
"Excess H2" is available for ethanol production when the ratio of the
moles
of H2 in the feed gas to the sum of two times the moles of CO converted
and three
times the moles of CO2 converted is greater than 1Ø If this ratio is
less than 1.0,
excess H2 is not available and ethanol can only be produced through a
different
controlling mechanism.
The term "fermentation" means fermentation of CO to alcohols and acetate. A
number of anaerobic bacteria are known to be capable of carrying out the
fermentation of
CO to alcohols, including 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
lungdahlii, including those described in WO 00/68407, EP 117309, US patent nos
5,173,429, 5,593,886, and 6,368,819, WO 98/00558 and WO 02/08438, and
Clostridium
autoethanogenum (Aribini 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). The disclosures of each of these
publications are
fully incorporated herein by reference. In addition, other acetogenic
anaerobic bacteria
may be selected for use in the process of the invention by a person of skill
in the art. It
will also be appreciated that a mixed culture of two or more bacteria may be
used in the
process of the present invention. One micro-organism suitable for use in the
present
invention is Clostridium autoethanogenum that is available commercially from
DSMZ
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and having the identifying characteristics of DSMZ deposit number DSMZ 10061.
The
fermentation may be carried out in any suitable bioreactor, such as a
continuous stirred
tank reactor (CTSR), a bubble column reactor (BCR) or a trickle bed reactor
(TBR).
Also, in some preferred embodiments of the invention, the bioreactof 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 (ethanol and acetate) is produced.
The term "gaseous substrates" as used herein means CO alone, CO and H2,
CO2 and H2, or CO, CO2 and H2, optionally mixed with other
elements or compounds, including nitrogen and methane in a gaseous state. Such
gaseous substrates include gases or streams, which are typically released or
exhausted to
the atmosphere either directly or through combustion. In some embodiments of
this
method the gaseous substrate comprises CO. In other embodiments of this
method, the
gaseous substrate comprises CO2 and H2. In still other embodiments,
the
gaseous substrate comprises CO and H2. In a particularly preferred
embodiment, the
gaseous substrate comprises CO, CO2 and H2. Still other substrates
of the
invention may include those components mentioned above and at least one gas of
nitrogen, CO2, ethane and methane. Thus, such substrates include what is
conventionally referred to as "syngas" or synthesis gas from the gasification
of carbon
products (including methane), as well as waste gases from a variety of
industrial
methods.
The phrase "high concentration of ethanol" means greater than about 10 g/L,
preferably greater than 15 g/L ethanol in fermentation broth or a product
ratio of ethanol
to acetate of 5:1 or more.
The terms "limiting substrate" or "limiting nutrient" define a substance in
the
nutrient medium or gaseous substrate which, during bacterial culture growth in
the
bioreactor, is depleted by the culture to a level which no longer supports
steady state or
stable bacterial growth in the bioreactor. All other substances in the
nutrient medium or
gas substrate are thus present in excess, and are "non-limiting". The evidence
for
limitation is that an increase in the rate of addition of the limiting
substrate, i.e. in the
nutrient feed rate or gas feed rate, to the culture causes a corresponding
increase in the
rate of gas uptake (mmol/min of gas) due to increase in cell density.
The term "microorganism" includes bacteria, fungi, archaea, and protists;
microscopic plants (called green algae); and animals such as plankton, the
planarian and
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the amoeba. Some also include viruses, but others consider these as non-
living.
Microorganisms live in all parts of the biosphere where there is liquid water,
including
soil, hot springs, on the ocean floor, high in the atmosphere and deep inside
rocks within
the Earth's crust. Microorganisms are critical to nutrient recycling in
ecosystems as they
act as decomposers. Microbes are also exploited by people in biotechnology,
both in
traditional food and beverage preparation, and in modern technologies based on
genetic
engineering. It is envisioned that mixed strain microorganisms, that may or
may not
contain strains of various microorganisms, will be utilized in the present
invention. In is
further envisioned that recombinant DNA technology can create microorganisms
using
select strains of existing microorganisms. In some embodiments of the present
invention, several exemplary strains of C. ljungdahlii include strain PETC
(U.S. Pat. No.
5,173,429); strain ERI2 (U.S. Pat. No. 5,593,886) and strains C-01 and 0-52
(U.S. Pat.
No. 6,136,577). These strains are each deposited in the American Type Culture
Collection, 10801 University Boulevard, Manassas, Va. 20110-2209, under
Accession
Nos.: 55383 (formerly ATCC No. 49587), 55380, 55988, and 55989 respectively.
Each
of the strains of C. ljungdahlii is an anaerobic, gram-positive bacterium with
a guanine
and cytosine (G+C) nucleotide content of about 22 mole %. These bacteria use a
variety
of substrates for growth, but not methanol or lactate. These strains differ in
their CO
tolerance, specific gas uptake rates and specific productivities. In the
"wild" strains found
in nature, very little ethanol production is noted. Strains of C. ljungdahlii
operate ideally
at 37° C., and typically produce an ethanol to acetyl (i.e. which
refers to both free
or molecular acetic acid and acetate salts) product ratio of about 1:20 (1
part ethanol per
20 parts acetyl) in the "wild" state. Ethanol concentrations are typically
only 1-2 g/L.
While this ability to produce ethanol is of interest, because of low ethanol
productivity
the "wild" bacteria cannot be used to economically produce ethanol on a
commercial
basis. With minor nutrient manipulation the above-mentioned C. ljungdahlii
strains have
been used to produce ethanol and acetyl with a product ratio of 1:1 (equal
parts ethanol
and acetyl), but the ethanol concentration is less than 10 g/L, a level that
results in low
productivity, below 10 g/Lday. In addition culture stability is an issue,
primarily due to
the relatively high (8-10 g/L) concentration of acetyl (2.5-3 g/L molecular
acetic acid) in
combination with the presence of ethanol. Furthermore, as the gas rate is
increased in an
effort to produce more ethanol, the culture is inhibited, first by molecular
acetic acid and
then by CO. As a result, the culture becomes unstable and fails to uptake gas
and
produce additional product. Further, early work by the inventors showed
difficulty in
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producing more than a 2:1 ratio of ethanol to acetyl in a steady state
operation. See, e.g.,
Masson et al., 1990 Applied Biochemistry and Biotechnology, Proceedings of the
l lth Symposium on Biotechnology for Fuels and Chemicals, 24/25: 857;
Phillips et
al., 1993 Applied Biochemistry and Biotechnology, Proceedings of the 14th
Symposium on Biotechnology for Fuels and Chemicals, 39/40: 559, among others.
A
large number of documents describe the use of anaerobic bacteria, other than
C.
ljungdahlii, in the fermentation of sugars that do not consume CO, CO2
and H2
to produce solvents. In an attempt to provide high yields of ethanol, a
variety of
parameters have been altered which include: nutrient types, microorganism,
specific
addition of reducing agents, pH variations, and the addition of exogenous
gases. See,
e.g., Rothstein et al, 1986 J. Bacteriol., 165(1):319-320; Lovitt et al, 1988
J. Bacteriol.,
170(6):2809; Taherzadeh et al, 1996 Appl. Microbiol. Biotechnol., 46:176.
By the term "mixed strains," it is meant a mixed culture of two or more of the
microorganism. Such "mixed strains" of the microorganism enumerated
hereinabove are
utilized in the methods of this invention.
The term "natural state" describes any compound, element, or pathway having no
additional electrons or protons that are normally present. Conversely, the
term "reduction
state" describes any compound, element, or pathway having an excess of one or
more
electrons. The "reduction state" is achieved by adding one or more electrons
to the
"natural state", i.e. by lowering the redox potential of the fermentation
broth.
"Nutrient medium" is used generally to describe conventional bacterial growth
media which contain vitamins and minerals sufficient to permit growth of a
selected
subject bacteria. Sugars are not included in these media. Components of a
variety of
nutrient media suitable to the use of this invention are known and reported in
prior
publications, including those of the inventors. See, e.g. the nutrient media
formulae
described in International Patent Application No. W008100558; U.S. Pat. No.
5,807,722;
U.S. Pat. No. 5,593,886, and U.S. Pat. No. 5,821,111, as well as in the
publications
identified above. According to the present invention, a typical laboratory
nutrient
medium for acetate production from CO, CO2, and H2 contains 0.9 mg/L
calcium pantothenate. However, a typical laboratory nutrient medium for
ethanol
production from CO, CO2, and H2 contains 0.02 mg/L calcium
pantothenate.
The term "reducing gas" means either or both CO or H2. By the phrase "an
amount of reducing gas greater than that required for growth of the bacteria'
is mean that
amount of reducing gas that exceeds the amount that the bacteria can use for
growth or
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metabolism, given the nutrient medium ingredients. This amount can be achieved
by
increasing the net amount of reducing gas, or by reducing key nutrient
ingredients, so
that the excess amount of gas is achieved without increasing the gas, or by
increasing the
rate of gas delivery to the bacteria. When the bacteria are exposed to more
reducing gas
than required for growth, the bacteria respond by increasing the producing of
ethanol.
"Subject bacteria" are acetogenic anaerobic (or facultative) bacteria, which
are able to
convert CO and water or H2 and CO2 into ethanol and acetic acid
products.
Useful bacteria according to this invention include, without limitation,
Acetogenium
kivui, Acetobacterium woodii, Acetoanaerobium noterae, Clostridium aceticum,
Butyribacterium methylotrophicum, C. acetobutylicum, C. thermoaceticum,
Eubacterium
limosum, C. ljungdahlii PETC, C. ljungdahlii ERI2, C. ljungdahlii C-01, C.
ljungdahlii
0-52, and Peptostreptococcus productus. Other acetogenic anaerobic bacteria
are
selected for use in these methods by one of skill in the art.
The term "syngas" means synthesis gas which is the name given to a gas mixture
that contains varying amounts of carbon monoxide and hydrogen. Examples of
production methods include steam reforming of natural gas or liquid
hydrocarbons to
produce hydrogen, the gasification of coal and in some types of waste-to-
energy
gasification facilities. The name comes from their use as intermediates in
creating
synthetic natural gas (SNG) and for producing ammonia or methanol. Syngas is
also
used as an intermediate in producing synthetic petroleum for use as a fuel or
lubricant via
Fischer-Tropsch synthesis and previously the Mobil methanol to gasoline
process.
Syngas consists primarily of hydrogen, carbon monoxide, and very often some
carbon
dioxide, and has less than half the energy density of natural gas. Syngas is
combustible
and often used as a fuel source or as an intermediate for the production of
other
chemicals
Detailed Embodiments of the Present Invention
The present invention relates to methods for sustaining microorganism culture
in
a syngas fermentation reactor in decreased concentration or absence of various
substrates
comprising: adding carbon dioxide and optionally alcohol; maintaining free
acetic acid
concentration to less than 5 g/L free acetic acid; and performing the above
mentioned
steps within 0-30 minutes, within 0-15 minutes, within 15 -30 minutes.
The present invention further contemplates a method for preventing rapid loss
of
microorganism culture in a syngas fermentation reactor in decreased
concentration or
absence of various substrates comprising: adding carbon dioxide and optionally
alcohol;
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decreasing temperature from the operating temperature to between 0-25 degrees
C while
maintaining the temperature between 0-25 C; maintaining free acetic acid
concentration
to less than 5 g/L free acetic acid; and performing the above mentioned steps
within 0-30
minutes, within 0-15 minutes, within 15 -30 minutes.
The present invention further provides a method for sustaining microorganism
culture in a syngas fermentation reactor due to decreased concentration or
absence of
various substrates in feed gas supply comprising: adding carbon dioxide and
optionally
alcohol; decreasing temperature from operating temperature to between 0-25
degrees C.
while maintaining the temperature between 0-25 C; maintaining free acetic acid
concentration to less than 5 g/L free acetic acid; and performing the above
mentioned
steps within 0-30 minutes, within 0-15 minutes, within 15 -30 minutes.
As an embodiment, said sustaining microorganism culture comprises duration of
about 0-30 hours. As an embodiment, pH which can be maintained in the range of
about
3.5-5.6. It is further contemplated that a bicarbonate solution is added to
control pH.
Bicarbonate solution can comprise: ammonium bicarbonate, sodium bicarbonate,
and/or
potassium bicarbonate. An embodiment of the present invention provides a
method
wherein optionally removing said carbon dioxide into the said reactor.
Furthermore, as
an embodiment, optionally adding nutrients to said reactor is provided. The
present
invention provides optionally adding nutrients to said reactor.
Further embodiments of the present invention provide alcohol comprising one or
more of the following: ethanol, butanol, ethanol and butanol.
Optionally, the temperature can be decreased from the operating temperature to
between 0-25 degrees C while maintaining the temperature between 0-25 C;
optionally
water can be added to said reactor. This water can comprise fresh water, make-
up water,
recycle water, distilled water, deionized water or their combinations.
The present invention contemplates a method wherein said microorganism
culture contains at least one acetogenic bacteria. The microorganism culture
can
comprise one or more strains selected from Clostridium, Moorella, and
Carboxydothermus or their genetic modifications.
As an embodiment, the microorganism can comprise Clostridium ljungdahlii
selected from the strains consisting of PETC, ERI-2, 0-52 and C-01 or their
combinations.
The present invention also provides a method wherein microorganism culture is
returned to pre suspension conditions comprising addition of syngas.
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Optionally as embodiments, the present invention, can provide for: removing
permeate; purging said reactor with inert gas; or maintaining low agitation to
keep solids
in suspension.
Other aspects and advantages of the present invention are described further in
the
following detailed description.
Acetogenic autotrophic bacteria that utilize carbon monoxide and/or hydrogen
and carbon dioxide (synthesis gas) to produce alcohol require a constant
supply of the
gas to produce alcohol. An interim product in the production of ethanol is
acetic acid,
which may be intercellular and extracellular. Without a sufficient synthesis
gas supply,
limited alcohol is produced in favor of acetic acid.
During conditions when there is reduced or no synthesis gas available for
production of the interim product, acetic acid, the culture can convert
alcohol back to
acetic acid in the presence of carbon dioxide. Ethanol is already present in
the culture
broth and is readily available in the event of limited or no synthesis gas.
Additional
alcohol could also be supplied as needed. Carbon dioxide can be added by
bubbling the
CO2 gas into the culture or it can be formed in the culture broth by the
addition of
bicarbonate. Sodium bicarbonate can be used in the fermentation to maintain
the desired
pH and is therefore readily available. In the acidic culture broth the
bicarbonate buffer
reacts to form carbon dioxide. The formed carbon dioxide is then available to
the
bacterium to shift alcohol back to acetic acid.
The shift of alcohol to free acetic acid in the presence of carbon dioxide is
a
relatively fast process. Microorganisms such as Clostridium ljungdahlii are
limited in the
concentration of free acetic acid that can be withstood in the culture broth.
Steps need to
be taken to control the concentration of free acetic acid during the reduction
or loss of
synthesis gas. One such method of control is with temperature manipulation.
Increased
temperature, within the mesophilic range, increases culture activity rates.
Whereas
reduced temperature in the fermentation broth reduces those rates. Therefore
reducing
temperature is helpful in retarding the activity of the culture during reduced
or no gas
conditions, resulting in a slower acid production. Another method of free
acetic acid
control is changing the culture pH. The equilibrium of acetyl to acetic acid
is controlled
in part by pH. Raising the pH during synthesis gas supply interruption permits
the total
acetyl concentration, acetyl plus acetic acid, to be higher while maintaining
a lower free
acid concentration.
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A third method with the potential for controlling the free acid concentration
is an
increased liquid flow through the system. As the free acid concentration
increases,
increasing the flow of a liquid stream into the system with an increase in the
permeate
purge will wash more free acid out of the culture while preventing unwanted
cell
washout. The additional liquid into the system may be an additional water
stream or an
increase in the flow of the nutrient supply stream.
DETAILED DESCRIPTION OF THE PROCESS UNDER NORMAL OPERATING
CONDITIONS
The present invention involves methods for the anaerobic fermentation of
gaseous substrates containing at least one reducing gas, particularly the
gaseous
components of industrial waste and synthesis gases (e.g., CO, CO2 and
H2) to
ethanol. These methods yield ethanol productivities greater than 10 g/Lday by
manipulating the biological pathways of the subject bacteria. One method of
the
invention causes an abundance of NAD(P)H over NAD(P). The oxidation of NAD(P)H
to NAD(P) causes acetic acid produced by the culture to be reduced to ethanol.
Alternatively, other methods for the production of high concentrations of
ethanol in an
anaerobic fermentation of this invention involve reducing the redox potential
of the
fermentation broth, and thereby reducing acetic acid to ethanol. The methods
of this
invention produce high ethanol concentrations (i.e., greater than about 10
g/L, and
preferably greater than about 15 g/L) and low acetate concentrations (i.e.
less than about
5 g/L free acetic acid in the bioreactor). These methods also maintain and
control method
conditions for continuous ethanol and acetic acid production to help the
system recover
rapidly from method upsets. Further, the methods of this invention help
prevent culture
acclimation to low nutrient concentration, which can be detrimental to culture
performance. The present invention provides a viable commercial method for
ethanol
production.
The Biological Pathways Utilized in the Method of this Invention Under Normal
Operating Conditions.
Without wishing to be bound by theory, the inventors theorize that the methods
for increasing the anaerobic production of ethanol from the methods described
herein are
based upon the biological pathways involving the conversion of NAD(P)H to
NAD(P) in
the basic pathway cycles of the acetogenic pathway for autotrophic growth. The
invention involves manipulating those pathways to enable continuous production
and
maintenance of high concentrations of ethanol with low acetate concentrations
under
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stable operating conditions, thereby providing commercially useful methods for
ethanol
production from industrial gases. The essential involvement of NAD(P)H to
NAD(P) in
the biological pathways is described as follows: The production of ethanol
from gaseous
components, such as CO, CO2, and H2 occurs in a three step
biological
method. In the first step, the substrates CO and H2 are oxidized and, in
doing so,
release NAD(P)H: NAD(P). fwdarw.NAD(P)H
CO+H2+H. sub. 20. fwdarw. CO. sub. 2+4H. sup.+
The products of step 1 are then converted to acetic acid, a step that requires
NAD(P)H: NAD(P)H.fwdarw.NAD(P) CO+CO2+6
H+.fwdarw.CH 3000H+H20 Finally, if excess NAD(P)H is available
because the reaction of step 1 proceeds at a faster rate than the reaction of
step 2, acetic
acid is reduced to ethanol. NAD(P)H.fwdarw.NAD(P) CH3COOH+4
H. sup.+. fwdarw.C. sub. 2H 50H+H. sub.20 Thus, the availability of
excess NAD(P)H
from substrate oxidation leads to the production of ethanol from acetic acid.
There are two known basic pathway cycles in the acetogenic pathway: (1) the
Acetyl-CoA cycle and (2) the THE cycle, in which CO2 is reduced to a
methyl
group. The sequence for the generation of ethanol and acetic acid therefrom is
illustrated
in J. R. Phillips et al, 1994 Applied Biochemistry and Biotechnology,
45/46:145. The
Acetyl-CoA cycle has an inner cycle, referred to herein as the CO cycle. As
the CO cycle
normally reacts clockwise, ferredoxin is reduced. Ferredoxin can also be
reduced by
H2 as it is oxidized on the enzyme hydrogenase. As a result, the Acetyl-
CoA cycle
also reacts clockwise, and ferredoxin is oxidized. If the inner CO cycle and
the Acetyl-
CoA cycle react at the same rates, ferredoxin is in a redox-state equilibrium.
If however,
these two cycles do not occur at the same rate, i.e., the CO cycle reacts at a
faster rate
than the Acetyl-CoA cycle, reduced ferredoxin is built up. Also with excess
H2,
reduced ferredoxin can also be produced in excess. This excess reduced
ferredoxin
causes the NAD(P) to be regenerated (reduced) to NAD(P)H, which builds an
excess that
must be relieved to equilibrium and in doing so, reduces acetic acid to
ethanol.
The THE cycle functions for cell growth and is necessary for a continuous
culture; therefore it cannot be completely stopped. Reducing the THE cycle
rate also
serves to cause a higher NAD(P)H to NAD(P) ratio. NAD(P)H is oxidized in two
places.
By limiting this oxidation, which would keep the total cellular NAD(P)H to
NAD(P)
ratio in balance, the NAD(P)H is used to reduce acetic acid to ethanol.
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A second basic method of causing acetic acid to be reduced to ethanol is by
directly lowering the redox potential of the fermentation broth. A reduction
state
sufficiently lower than the natural state of the culture causes NAD(P)H to be
in
abundance and promote the reduction of acetic acid to ethanol.
The Methods of the Normal Operation
The basic steps of the method include the following: A continuous fermentation
method with product recovery is described by reference to FIG. 1. A continuous
flow of
gaseous substrate 1 comprising at least one reducing gas, e.g., CO or H2,
is supplied
at a selected gas feed rate and a continuous flow of liquid phase nutrient
medium 2 at a
selected nutrient feed rate are supplied to a fermentation bioreactor 3
containing a subject
bacteria. In the bioreactor 3, the medium and gaseous substrate are fermented
by the
bacteria to produce ethanol and acetate acid. Once a stable cell concentration
is achieved
under steady state conditions, the components of the continuous system are
manipulated
to reduce the redox potential, or increase the NAD(P)H to NAD(P) ratio, in the
fermentation broth, while keeping the free acetic acid concentration in the
bioreactor less
than 5 g/L. The methods of this invention are designed to permit and maintain
production
of ethanol and acetate in the fermentation broth such that the ethanol
productivity is
greater than 10 g/Lday at an ethanol to acetate ratio of between 1:1 and 20:1.
In one
embodiment, that ratio is greater than 3:1. In another embodiment, that ratio
is greater
than 5:1. In still another embodiment, that ratio is greater than 10:1. In
still another
embodiment that ratio is greater than 15:1. The method of this invention is
alternatively
effective in enhancing stable continuous (steady state) production of high
ethanol
concentrations (15-35 g/L ethanol) and low acetate concentrations (0-5 g/L
acetate), i.e.,
ethanol to acetate product ratio of 3:1 or more, from CO, CO2, and
H2 with
good method stability.
Periodically, during the course of the methods of this invention, samples of
the
broth are removed to determine the ratio by a conventional assay method. For
example,
the cells are separated from the sample, e.g., by centrifugation and the cell-
free sample is
then subject to an assay method, such as the preferred method of gas
chromatography.
However, other conventional assay methods are selected by one of skill in the
art. The
additional optional steps of the method are added to achieve and/or maintain
the ratio.
Steps used to manipulate the system components and maintain and/or achieve the
desired ethanol productivity or the ethanol to acetate ratio include at least
one, and
desirably, combinations of the following steps: altering nutrient medium
contents,
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nutrient feed rate, aqueous feed rate, operating pressure, operating pH,
gaseous substrate
contents, gas feed rate, fermentation broth agitation rate, avoiding product
inhibition
step, decreasing cell density in the bioreactor, or preventing substrate
inhibition. Some
preferred manipulations include supplying the bioreactor with liquid phase
nutrient
(pantothenate or cobalt) limitation, a slight excess of CO and H2 in the
feed gas,
minimizing acetate concentration, avoiding culture acclimation to low liquid
phase
nutrient concentrations, bringing the culture to a suitable cell concentration
at a relatively
fast rate, raising the pH of the culture above 4.5, purging bacterial cells
from the
bioreactor to a cell concentration less than the stable steady state
concentration that
utilizes all reducing gas or nutrient substrates in the bioreactor and
increasing the
aqueous feed rate when the free acetic acid portion of the acetate present in
the
fermentation bioreactor broth exceeds 2 g/L, thereby inhibiting any unwanted
increase in
the concentration of free acetic acid. All of these steps are described in
detail below.
Exhaust gas 4 containing gases other than CO, CO2 and H2 and
unconverted CO, CO2 and H2 from the reactor are vented from the
reactor and
are used for their fuel value. If excess H2 as a controlling mechanism is
employed,
the H2 partial pressure in the outlet gas and ratio of H2 partial
pressure to
CO2 partial pressure in the exit gas are used to identify the control of
the ethanol to
acetate ratio by that step. Cell recycle is used (but is not required) to
increase the
concentration of cells inside the bioreactor, and thus provide more
biocatalyst for CO,
CO2 and H2 conversion. With cell recycle, liquid effluent from the
reactor 5 is
sent to a cell separator 6 where the cells 7 and permeate (cell free liquid) 8
are separated.
The cells 7 are sent back to the bioreactor and the permeate 8 is sent to
product recovery.
Cell separation is accomplished by using a continuous centrifuge, hollow fiber
or
spiral wound filtration system, ceramic filter system or other solid/liquid
separator.
Ethanol can be recovered from the permeate (or alternatively the effluent from
the
reactor 5 if cell separation is not employed) by a variety of techniques
including
distillation and adsorption. Permeate 8 is separated in a distillation column
to produce
95% ethanol overhead 10, and water 11 for recycle back to the reactor 3. The
recycle
water 11 contains excess nutrients not used in the fermentation, but any
excess vitamins
from fermentation or cell lysis are destroyed by thermal distillation. The 95%
ethanol
overhead 10 is sent to a molecular sieve 12 where anhydrous ethanol 13, the
desired final
product, is separated from dilute ethanol 14 which is sent back to the
distillation column
9.
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The continuous combination of growth, death and cell purge maintains a
constant
cell concentration, such that a continuous method used in producing ethanol
(and small
amounts of acetic acid) can operate for many months by being fed CO, CO2
and
H2 along with nutrients without additional culture supplementation. The
methods of
this invention maintain and control conditions for continuous ethanol and
acetic acid
production and prevent or correct rapidly for method upsets. The methods of
this
invention also help prevent culture acclimation to low nutrient concentration,
which can
be detrimental to culture performance. In the descriptions below and in the
examples,
unless otherwise indicated, the pressure used is 1 atmosphere and the
temperature used is
between 36-41° C. Desirable temperatures and pressures may be
determined by
one of skill in the art, depending on the microorganism selected for use in
the bioreactor.
A variety of manipulations, described specifically below, added to the basic
steps
of this invention permit the enhanced production of ethanol. Preferably,
liquid phase
nutrient limitation (pantothenate or cobalt) or the use of excess H2 or
CO are the
method steps of the invention, described in detail below, used to achieve and
maintain
the desired ethanol productivity and permit production of stable
concentrations and ratios
of ethanol to acetate in the fermentation broth. These conditions permit
production of
stable concentrations of ethanol and acetate in the fermentation broth. In a
preferred
embodiment, the ethanol to acetate product ratio produced in the fermentation
broth is
greater than 10:1 and the ethanol concentration is greater than 15 g/L.
A. Calcium Pantothenate Limitation
In one specific embodiment of this invention, the method for manipulating the
biological pathways to favor ethanol production and limit acetic acid
production involves
limiting the amount of calcium pantothenate in the nutrient medium to an
amount which
is less than required to maintain the bacteria at a stable, steady state
concentration that
would fully utilize the calcium pantothenate provided. Pantothenate is a
component of
Acetyl-CoA and therefore, by limiting calcium pantothenate in the nutrient
medium, the
Acetyl-CoA cycle rate is reduced relative to the CO cycle rate. This causes a
build-up of
reduced ferredoxin and the reduction of NAD(P) to NAD(P)H, and thereby
increases the
production of ethanol as the final product.
Pantothenate limitation is observed when the micrograms (µg) of calcium
pantothenate fed to the reactor per gram (g) of cells (dry weight) produced in
the reactor
is in the range of 0.5 to 100. A more desirable pantothenate limitation is in
the range of 2
to 75 mu.g of calcium pantothenate per gram (g) of dry cells produced in the
reactor.
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Still a preferred pantothenate limitation is in the range of 0.5 to 50 µg
of calcium
pantothenate per gram (g) of cells produced in the reactor. Another embodiment
of this
limitation is at about 1-25 mu.g of calcium pantothenate per grain (g) of
cells produced
in the reactor. Another embodiment of this limitation is at about 10-30 mu.g
of calcium
pantothenate per gram (g) of cells produced in the reactor. This amount of the
nutrient
maintains ethanol production in preference to acetate production.
In another aspect of this method, the acclimation of the bacteria in the
fermentation bioreactor to low limiting calcium pantothenate concentration is
avoided by
regulating or adjusting the fermentation parameters, so that a constant
calcium
pantothenate concentration is maintained, while at least one, and sometimes
more than
one, parameter of gas feed rate, liquid feed rate, agitation rate, or H2
partial pressure
is adjusted. Major changes in nutrients are avoided, but a relatively constant
nutrient feed
concentration is maintained. If the culture is allowed to acclimate to low
liquid phase
limiting nutrients, poor product ratios of 1.0 g ethanol/g acetate or less
occurs in an
irreversible method. Thus, reactor shut down and reinoculation is necessary.
Preferably,
the biological pathway is controlled to favor ethanol production and limit
acetic acid
production by first supplying excess H2 in the feed gas to the
bioreactor, and then
limiting calcium pantothenate in the nutrient medium as described above.
In fact, at start-up, the normally limiting liquid phase nutrient calcium
pantothenate is kept in excess to avoid acclimation to low nutrient
concentrations, a
condition that can result in very poor performance and the loss of the
culture's ability to
produce achieve high ethanol productivities of more than 10 g/Lday if excess
H2 is
not employed.
B. Cobalt Limitation
In another embodiment of this invention, the method for manipulating the
biological pathways to favor ethanol production and limit acetic acid
production involves
limiting the amount of cobalt in the nutrient medium to an amount which is
less than
required to maintain the bacteria at a stable steady state concentration that
would fully
utilize the cobalt provided. Cobalt limitation is observed when the micrograms
(µg) of
cobalt fed to the reactor per gram (g) of cells (dry weight) produced in the
bioreactor is
in the range of 5 to 100. Preferably, a cobalt limitation involves providing
between about
20 to 50 mu.g of cobalt to the reactor per gram of cells produced in the
reactor. This
amount of cobalt maintains ethanol production in preference to acetate in the
process.
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Limiting cobalt in the fermentation broth may also reduce the Acetyl-CoA cycle
rate. Because cobalt is used to transfer a methyl group from the THE cycle to
the Acetyl-
CoA cycle, limiting the amount of cobalt in the fermentation broth also
reduces the THE
cycle function by not permitting the transfer. Cobalt limitation reduces the
THE cycle
rate, which also causes a higher NAD(P)H to NAD(P) ratio, thereby producing
ethanol.
The method is further manipulated by preventing acclimation to low limiting
cobalt concentration. In much the same manner as acclimation to low
pantothenate
concentrations is avoided, a constant cobalt concentration is maintained while
adjusting
one or more of the fermentation parameters (gas rate, liquid rate, agitation
rate, CO2
content, and H2 gas partial pressure). Major changes in nutrients is
avoided, but
instead a relatively constant nutrient feed concentration is maintained.
Preferably, the biological pathway is controlled to favor ethanol production
and
limit acetic acid production by first feeding excess H2 to the reactor
and then
limiting cobalt in the nutrient medium as described above. At start-up, the
limiting liquid
phase nutrient cobalt is kept in excess to avoid acclimation to low nutrients
concentration, a condition that can result in very poor culture performance
and the loss of
the culture's ability to produce product ratios greater than 1:1.
C. Oversupplying Hydrogen
In still another embodiment, the method for manipulating the biological
pathways
to favor ethanol production and limit acetic acid production involves feeding
excess
H2 in the feed gas or limiting gaseous carbon which results in excess
H2, which
is then used by the biological pathway. Preferably, the H2 reducing gas
is in excess
relative to CO, and the excess H2 causes the bacteria to produce a high
ethanol to
acetate ratio in the fermentation broth. If the ratio of the H2 (moles of
gas fed) to the
sum of two times the CO (in moles of gas) converted and three times the
CO2 (in
moles of gas) converted is greater than 1, the fermenter is carbon limited.
The H2
partial present in the exit gas is preferably greater than 0.4 atm. Finally
the ratio of
H2 partial pressure to CO2 partial pressure must be greater than 3.0
to assure
that sufficient H2 is available to use all the CO2. If the CO2
partial
pressure is greater than 0.1 atm, it is likely that growth.has been otherwise
limited.
During start-up, the use of excess H2 is favored over nutrient
limitation,
mainly because it is easier to control. The benefits of employing excess
H2 are that
it avoids excess acetic acid production, which can lead to poor product ratios
and
potential acetic acid inhibition, as well as acclimation to low nutrient
concentrations.
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D. Oversupplying Carbon Monoxide
Another way of manipulating the components of the method involves
oversupplying the reducing gas, CO, in the gaseous substrate for use in the
pathway,
which serves to directly lower the redox potential in the fermentation broth.
Thus,
according to this embodiment, the bioreactor is suppled with gaseous substrate
comprising CO where the amount of CO present in the bioreactor is greater than
the
amount required to maintain the bacteria at a stable, steady state
concentration that would
fully utilized the CO provided. CO oversupply as a method of favoring ethanol
production over acetic acid production when the specific rate of CO uptake
(millimoles
of CO per gram of cells- (dry weight) in the reactor per minute, or mmol/g
cellmin) is
greater than 0.3. More preferably, this step involves a specific rate of CO
uptake of
greater than 0.5. This means that each cell on the average is utilizing CO in
its
metabolism at a rate of at least 0.3 mmol/gmin., or more ideally at a rate of
at least 0.5
mmol/gmin. Preferably, the CO is provided at a rate at which the CO uptake is
from 0.3
to 2 mmol CO/gram cell (dry weight) of bacteria/minute. In another embodiment,
the CO
is provided at a rate of from 0.5 to 1.5 mmol CO/gram cell (dry weight) of
bacteria/minute. In another embodiment, the CO is provided at a rate of about
1 mmol
CO/gram cell (dry weight) of bacteria/minute.
This rate of CO uptake maintains ethanol production in preference to acetate
production. If CO is supplied such that the dissolved CO in the fermentation
broth is
significant by gas pressure or extremely good mass transfer, the fermentation
broth
becomes more reduced. Oversupply of CO has two additional benefits. Excess CO
may
cause the CO cycle to operate at a faster rate, and if the Acetyl-CoA cycle is
otherwise
limited and cannot keep up with the CO cycle, reduced ferredoxin builds-up. CO
may
also slow down step 2 (production of the intermediate acetic acid) in the
overall three-
step method through substrate inhibition. This decreased rate of step 2 in
relation to step
1 causes an excess of NAD(P)H, which leads to ethanol production in favor of
acetic
acid.
Although excess CO can result in increased ethanol production by directly
reducing the redox potential of the fermentation broth, the presence of excess
CO also
inhibits growth by inhibiting the CO-dehydrogenase and therefore the uptake of
H2.
The presence of excess CO unfortunately also results in poor H2
conversion, which
may not be economically favorable. The consequence of extended operation under
substrate inhibition is poor H2 uptake. This eventually causes cell lysis
and
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necessary restarting of the reactor. Where this method has an unintended
result of CO
substrate inhibition (the presence of too much CO for the available cells)
during the
initial growth of the culture or thereafter, the gas feed rate and/or
agitation rate is reduced
until the substrate inhibition is relieved.
E. Additional Manipulating Steps
In addition to the major method enhancing steps described above, several
method
steps are desirably included in the ethanol production method.
1. Increasing Mass Transfer
One such additional embodiment involves ensuring that the mass transfer of the
CO or H2 from the gas feed to the liquid fermentation broth is faster
than the ability
of the bacteria to utilize the dissolved gases. For example, if a bioreactor
containing C.
ljungdahlii is fed CO, CO2 and H2 and is operated without limitation
on
nutrients (such as pantothenate or cobalt) or the presence of excess H2,
cell growth
is limited by the amount of gas transferred into the liquid phase and the
system produces
acetic acid as the product. If the culture is fed a slight amount of CO or
H2 in excess
of that required for culture growth, it produces ethanol. However, if too much
gas is
transferred into the liquid phase for the culture to use, substrate inhibition
occurs, which
can lead to culture upset and cell death. Thus, there is a very narrow range
of operation
with excess mass transfer.
With reference to the Acetyl-CoA cycle, in order for the excess reduced
ferredoxin to be produced, the CO cycle or the reduction of ferredoxin through
hydrogenase must occur faster than the Acetyl-CoA cycle. The methods described
herein
limit the rate at which the organisms can utilize the dissolved gases by
restricting the rate
at which essential nutrients e.g., calcium pantothenate or cobalt, or other
substrates, such
as CO2 gas, are available to the bacteria, or by providing excess
substrate, H2
or CO to the culture.
A theoretical rate of mass transfer, which is faster than the rate at which
the
bacteria can use substrate, even without other limitations, can be calculated.
That rate,
when achieved, is limited by the natural growth rate of the organism.
Therefore, the most
productive embodiment is where the mass transfer (gas flow rate or agitation
rate) is
faster than the rate at which the highest possible concentration of cells can
utilize the
substrate without any limitation. There would be a very narrow operating range
since
substrate inhibition could quickly cause cell death and a resulting by-product
concentration which is toxic to the culture.
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2. Supplying Excess CO and H2
In another embodiment of a method of this invention, stability in the high
ethanol
concentration/limited acetic acid production is achieved in the methods which
limit
cobalt or calcium pantothenate, or provide an abundance of H2 or CO.
According to
this step, as the culture uses the gaseous substrates CO, H2 and CO2
as the
carbon and energy sources, CO and H2 are supplied in slight excess. A
slight excess
of CO and H2 is achieved by attaining steady operation and then gradually
increasing the gas feed rate and/or agitation rate (10% or less increments)
until the CO
and H2 conversions just start to decline. This is one means of avoiding
mass transfer
limitation, which favors acetic acid production, and supplying excess reduced
ferredoxin
in order to reduce NAD(P) to NAD(P)H and produce ethanol. If CO and H2
are not
supplied in slight excess, mass transfer limitation occurs, and the pathway is
balanced.
This results in poor ethanol to acetate product ratios (high acetate
concentrations). High
acetate concentrations can ultimately result in acetic acid inhibition, which
limits the
ability of the bacterium to take up H2 and can eventually lead to culture
failure.
Steps to avoid mass transfer limitation include an increase in the agitation
rate or
gas rate to transfer more CO and H2 into the liquid phase, and thus
return to the
presence of a slight excess CO and H2. If product inhibition occurs as a
result of
mass transfer limitation, it is necessary to increase the liquid feed rate to
clear the acetic
acid inhibition, by diluting to a lower resulting acetate concentration. Since
increasing
the medium feed rate would increase the mu.g pantothenate or cobalt/g-cell
produced,
this must be done only briefly or the excess pantothenate or cobalt must be
eliminated by
adjusting the medium concentration or increasing the water feed rate.
3. Conditioning Acetic Acid Product Inhibition
Where in the methods described above, acetic acid product inhibition can occur
if
too much molecular acetic acid, i.e., >2 g/L, accumulates in the bioreactor to
allow cell
growth and further ethanol production. Another manipulating step is used to
avoid
culture failure. One modification involves briefly increasing the liquid or
aqueous feed
rate to reduce the liquid phase concentration of inhibiting acetic acid to
lower than 2 g/L.
4. Water Recycle Step
Still another optional method step for maintaining a stable culture which
produces ethanol as the only product with no net acetic acid production in the
methods of
this invention involves adding water recycle from distillation back to the
fermentation
reactor. As was noted earlier, water (containing up to 5 g/L acetate) recycle
has the
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benefit of recycling the produced acetate back to the reactor so that no net
acetic acid is
produced. An equilibrium is thus established between the ethanol and acetate
in the
reactor. As a result, all CO, CO2 and H2 fed to the reactor and
converted to
products results in ethanol production, except for that used for culture
maintenance.
5. Reducing Cell Density
Still another manipulating step useful in the method is to initiate periodic
or
continuous purging of bacterial cells from the bioreactor to reduce the cell
concentration
in the bioreactor. This manipulation serves to reduce the cell concentration
to less than a
stable, steady state cell concentration that utilizes all reducing gas or
nutrient substrates
in the bioreactor. By thus, altering the cell density, the production of
ethanol is favored
over the production of acetate in the bioreactor.
6. Two Stage CSTR
One of the problems associated with ethanol production with medium limitation
is the ability or tendency of the culture to eventually adapt to the limiting
conditions and
not continue to produce ethanol after several months of operation. Instead
acetate iscome
eventually the dominant product. This acclimation to low limiting nutrient
concentrations
results in a culture which produces more acetic acid than ethanol (ethanol to
acetate
product ratio of 1.0 or less), and yields low ethanol concentrations
(sometimes as low as
1 g/L). Adaptation most likely occurs when the culture is not provided with
sufficient
nutrients during start-up, where growth rate is more important than ethanol
production
rate. Additionally, there is a danger that the culture can be acclimated to
low limiting
nutrient concentrations during steady state operation particularly as the
limiting nutrient
concentrations are adjusted downward to rid the reaction system of acetate.
To avoid this adaptation when using the pantothenate or cobalt limiting steps
above, instead of allowing the culture to grow with the available nutrients,
and the
danger mentioned above, another modification of the method can be employed. A
two-
stage CSTR system where primarily good culture growth occurs in the first
stage on a
slight excess of limiting nutrients (perhaps with accompanying acetic acid
production),
followed by a production stage where the culture from the first stage is now
limited by
the limiting nutrient and is used to produce high concentrations of ethanol,
is another
modification of the method. This modification enables the maintenance of a
stable
culture, which does not acclimate to reduced pantothenate or cobalt
concentrations. This
modification involves operating a two-stage CSTR, in which a growth reactor
(Stage 1)
to feed a production reactor (Stage 2) where the bulk of the ethanol
production occurs.
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The growth reactor is not operated with the nutrient limitation steps
described above, so
the culture is not as susceptible to acclimation to a limited condition.
According an embodiment of two-stage CSTR, the Growth Stage is operated at a
liquid retention time (LRT) of about 24 hours. The Growth Stage CSTR 1 is fed
enough
pantothenate or cobalt in the medium 2 to yield a healthy culture (and may
produce some
acetic acid as well). Thus, excess acetic acid is produced in the reactor, but
with
increased stability. This pantothenate or cobalt concentration is in excess of
what would
normally be fed to a single CSTR used to produce ethanol. The gas feed to this
reactor is
unconverted gas 3 from the Production Stage 4 and the liquid feed is fresh
medium 2.
The Growth Stage CSTR is operated without cell recycle. The purpose of this
Growth
Stage reactor is to provide a healthy culture for later ethanol production
that does not
acclimate to low pantothenate concentrations.
The Production stage reactor 4 is operated at a nominal LRT of less than 20
hours. This CSTR with cell recycle is fed a fresh gas feed 5, and may have low
conversions. It is fed fresh medium feed 6 as well as culture feed 7 from the
Growth
Stage. Minimal pantothenate or cobalt is fed to this reactor since the excess
from the
Growth Stage is available. Cell recycle 8 is used in this reactor in order to
get the most
production out of the cells sent back to the reactor 9. The exit ethanol
concentration in
the liquid product 10 should be greater than 20 g/L. The features of the two-
stage CSTR
system include little change for acclimation to low pantothenate or cobalt
concentrations;
an overall LRT of less than or equal to 30 hours; an expected greater ethanol
productivity
and higher ethanol concentration than from a single CSTR of the same size.
7. Start-up Modifications
Still other method steps, which are preferably utilized in the practice of
this
invention, involve cell production in the initial start-up of the fermentation
culture. The
start-up of a bioreactor fed CO, CO2 and H2 to produce ethanol and
acetic acid
is accomplished by batch inoculation from stock culture or by employing a
continuous
inoculum from an existing reactor as culture feed. As noted earlier in the
discussion of
avoiding culture acclimation to low pantothenate or cobalt concentrations, the
culture is
most desirably brought up to a high cell concentration prior to limiting
nutrients, but
supplying excess H2 to the culture. This rapid start-up avoids culture
acclimation
and yields good product ratios (high ethanol and low acetate concentrations).
If the rapid
start-up is not employed, poor product ratios can occur and the culture can
acclimate to
low liquid phase nutrient concentrations and require reactor reinoculation.
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The reactor is started with a batch liquid phase (liquid medium is not
initially fed
continuously to the reactor), at low agitation rates (perhaps 400-600 rpm in a
laboratory
New Brunswick Scientific Bioflo® reactor) and at the desired pH. The
liquid phase
in the reactor thus consists of a batch of nutrient medium containing vitamins
and salts,
with a nominal concentration of limiting nutrient, either calcium pantothenate
or cobalt
(20 µg/L pantothenate or 75 ppb cobalt). If continuous inoculum from an
existing
reactor is employed, batch liquid phase operation likely is not necessary. In
this case, gas
is fed continuously to the reactor during initial start-up at a slow rate.
Ideally, the gas
phase at start-up would be CO2-free, H2-abundant and the gas rate
and
agitation rate would be kept at low levels to avoid CO substrate inhibition.
An exemplary general start-up protocol for producing and sustaining
commercially viable ethanol concentrations from CO, CO2 and H2
consists of
three distinct phases: (a) initial start-up, where cell production is
critical; (b) start-up
where production rate becomes critical; and (c) steady state operation.
Essentially, initial
start-up is characterized by inoculation of a batch liquid, with a nominal
limiting
nutrient, selected from cobalt (75 ppb) or calcium pantothenate (20 µg/L)
at a desired
pH (typically 4.5-5.5). To facilitate start-up, the gas feed rate and
agitation rate are
preferentially kept low, while H2 is fed in excess. The cause of ethanol
production
during start-up is excess H2; nutrient limitation occurs later. Thus,
excess liquid
nutrients are actually present during start-up to avoid unwanted culture
acclimation to
low nutrients. As the fermentation proceeds over a period of several hours
after
inoculation, CO2 is produced and H2 is consumed. The changes in
these rates
indicated that the agitation rate should be nominally increased slowly
(perhaps by 200-
300 rpm in a laboratory reactor, over a period of 2-3 days) to avoid mass
transfer
limitation.
This onset of CO2 production occurs much more rapidly in systems
employing continuous inoculation as opposed to batch inoculation from stock
culture.
However, if the agitation rate is increased too fast, CO substrate inhibition
occurs. This
procedure of watching H2 conversion (or CO2 production) while
nominally
increasing agitation rate occurs at a relatively rapid rate until the target
agitation rate is
reached. During this time of increasing agitation rate in batch liquid
culture, cell
production instead of product formation is of utmost importance.
Once the target agitation rate is reached (800-1000 rpm in laboratory New
Brunswick Scientific Bioflo® reactor), the culture is allowed to steady to
confirm
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H2 uptake. The start-up shifts to a mode in which production rate becomes
important. It is desirable to have CO conversions exceeding 80% and a high
H2
partial pressure in the exit gas (at least 0.55 atm) to assure ethanol
production while
limiting acetate and the free molecular acetic acid concentration. The liquid
medium feed
rate is then turned on (for systems having batch inoculation from stock
culture) to initiate
continuous liquid feed and the gas rate is increased in 10% increments toward
the target
flow rate. H2 remains in excess to avoid excess acetic acid production.
As the gas
rate is increased, the liquid phase nutrients are limited (calcium
pantothenate or cobalt),
and the effect of such limitation is a small drop in H2 conversion, at
the target
production.
At steady state operation, production of 15-35 g/L ethanol and 0-5 g/L acetate
is
reached. At this stage, small adjustments in limiting nutrients, liquid feed
rates and gas
feed rates are needed, and are chosen by one of skill in the art with resort
to knowledge
extant in the art as well as the teachings of this invention. If cell recycle
is to be added to
the method of ethanol production, it is added at this time along with an
adjustment in gas
rate (increase) and nutrient concentration (decrease).
The above described methods of continuously producing and maintaining high
concentrations of ethanol with low by-product acetate concentrations under
stable
operating conditions enhance the use of the subject bacteria on a commercial
scale for
ethanol production. The steps outlined in the methods above overcome the
limitations of
utilizing the subject bacteria for commercial ethanol production from CO,
CO2 and
H2. Preferably the method employs a continuous bioreactor, although batch
and fed-
batch fermentation methods are also used, but are not likely to be
economically viable
for large-scale ethanol production.
The following examples will serve to illustrate certain specific embodiments
of
the inventions herein disclosed. These Examples should not, however, be
construed as
limiting the scope of the novel invention as there are many variations which
may be
made thereon without departing from the spirit of the disclosed invention, as
those of
skill in the art will recognize.
EXAMPLES
An initial experiment was conducted to investigate using ethanol and carbon
dioxide as the energy source for maintaining the viability of C. Ljungdahlii.
In this
experiment the carbon dioxide was provided as a gas bubbled through the
culture. The
free acid concentration was controlled by lowering the temperature to 25
degrees C and
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by increasing the pH set point. The synthesis gas was turned off and replaced
with a slow
bubbling of carbon dioxide at approximately 30m1/min. Agitation was decreased
to a low level that provided just enough mixing to distribute heat and liquid
additions
into the reactor. The pH was raised from 4.5 to 4.7. The reactor had a cell
recycle loop
using a hollow fiber membrane, which permitted a permeate purge to be used in
order to
prevent cell loss during the experiment. The permeate flow was equal to the
flow of
medium into the system. The liquid retention time did not change, remaining at
30 hours.
After 12 hours of no synthesis gas supply, the measured ethanol and total
acetyl
concentrations had changed as was expected. The ethanol level decreased from
24.0 to
12.8g/L while the total acetyl level increased from 4.2 to 10Ag/L. The
temperature set
point was returned to 38 C. As the culture was heating, the agitation was
increased to the
same level used prior to the experiment; the carbon dioxide was replaced with
synthesis
gas flow at 50 percent the flow rate used prior to the experiment.
The permeate purge was stopped. The culture was maintained in this condition
for 14 hours. During that time the carbon monoxide uptake remained stable and
the
hydrogen uptake steadily improved. Once the hydrogen uptake had improved
sufficiently, the gas flowrate was stepwise increased to reach the pre-
experimental
flowrate. Within 47.5 hours the synthesis gas flow rate had returned to pre-
experimental
rates. As the feed gas flow was increasing, the total acetyl concentration was
decreasing
and the ethanol concentration increased. The total acetyl concentration was
back down to
pre- experimental levels within 32 hours. The ethanol concentration reached
near pre-
experimental levels within 70.5 hours.
In this experiment the carbon dioxide was provided by a continuous flow of a
7.7% sodium bicarbonate solution into the culture. The temperature was reduced
to
25 C. Free acetic acid concentration was controlled by lowering the
temperature to 25 C,
increasing pH and by increasing the liquid flow through the culture. The
synthesis gas
was turned off and replaced with a continuous flow of 7.7% sodium bicarbonate.
In the
presence of an acidic environment, sodium bicarbonate degrades into a sodium
ion,
water and carbon dioxide thus providing the necessary carbon dioxide for the
conversion
of ethanol to free acid. Agitation was decreased to a low level that provided
just enough
mixing to distribute heat and liquid additions into the reactor. The pH set
point was not
controlled, but as the bicarbonate was added to the culture, the pH slowly
increased
throughout the experiment which helped control the concentration of free acid.
The
reactor had a cell recycle loop using a hollow fiber membrane, which permitted
a
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permeate purge to be used in order to prevent cell loss during the experiment.
The
permeate flow was equal to the flow of medium plus the additional flow of
sodium
bicarbonate into the system. The extra bicarbonate flow reduced the liquid
retention time
from 29 to 21 hours.
During the experiment the ethanol concentration decreased as the total acetyl
concentration rose steadily. Within 5.5 hours the ethanol concentration had
decreased
from 21.0 to 14.1 g/L while the total acetyl level had increased from 4.4 to
9.1 g/L. The
measured pH had also increased from 4.48 to 4.84. In an effort to control the
acid
concentration, the nutrient stream flow rate was increased from 1.33mL/min to
2.81
mL/min., 5.6 hours after the start of the experiment. The permeate purge was
also
increased from 1.86 to 3.48mL/min to prevent unwanted cell washout. These
changes
decreased the liquid retention time from 21 to 12 hours. This had the desired
effect of
holding the acid concentration down. Two hours after the changes in liquid
flows the
total acetyl concentration had increased to only 9.4g/L. However, the ethanol
concentration dropped at a faster rate from 14.1 to 10. 7g/L. After 8 hours of
no synthesis
gas supply, the measured ethanol and total acetyl concentrations changed as
expected.
The ethanol level decreased from 21.0 to 10.7g/L while the total acetyl level
increased
from 4.4 to 9.4g/L. The temperature set point was raised back to 38 C. As the
culture
was heating, the agitation was increased to the same level used prior to the
experiment;
the sodium bicarbonate addition was stopped, synthesis gas flow was started at
50
percent the flow rate used prior to the experiment; and the permeate purge was
stopped.
The culture was maintained in that condition for only 50 minutes. The gas flow
rate was
stepwise increased to reach the prior flow rate. Within 29.2 hours the
synthesis gas flow
rate had returned to pre-experimental rates. As the feed gas flow was
increasing, the total
acetyl concentration decreased and the ethanol concentration increased to pre-
experimental concentrations within 43.2 hours. Thus ethanol that is already in
the
fermenter can thus be used along with carbon dioxide to maintain culture
viability during
synthesis gas interruption.
Example 1
Microorganism Gas Loss Studies Using Ethanol and C02 Conversion for Energy
The purpose of the microorganism experimentation was to determine a method of
sustaining culture in the event of a feed gas loss for an extended (>30
minute) period of
time. In this example, the focus was on C02 addition for the conversion of
ethanol to
free acid as a way for the culture to gain energy during the loss of synthesis
gas.
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It has been known for quite some time that certain acetogenic microorganisms
can convert ethanol back to acetic acid using C02, but no testing had been
done to
determine if this process could be used to sustain the culture for long
periods of time
when there was no synthesis gas available. An embodiment of the present
invention
provides a solution for surviving a loss of feed gas since ethanol and C02 (in
the form of
sodium bicarbonate) are readily available for use due to normal bioreactor
operations. In
addition to adding C02 for the conversion of ethanol to acid the culture
temperature was
decreased during some of the experiments as a way to slow culture activity. A
slower
cell activity should reduce the amount of energy needed, the amount of C02 and
ethanol
required, and the amount of acid produced.
For these experiments the bioreactor was run as a straight through CSTR with
both a cell recycle and culture cooling coil loop. A permeate purge was used
during the
experiments to prevent unwanted cell loss, but the purge stream was diverted
to waste
and was not recycled back into the bioreactor. During the normal bioreactor
operations
the culture temperature was kept 38 C; agitation was 400rpm; the approximate
culture
volume was about 2.4L; and the culture pH set point was 4.5. A solution of
7.7%
NaHCO3 was used for pH control. The feed gas was synthesis gas containing
15%H2,
45%N2, 30%CO and 10% C02. The syngas feed rate was about 475mL/min. Medium
was fed into the reactor at about 1.30-1.35mL/min, or about 1870-1940mL/day.
Liquid
and cell retention times averaged 25-30 hours. The medium used was the lx EtOH
medium regularly used for the C-01 culture. Medium components and their
concentrations are listed in Table 1 below.
During normal operations the CL bacteria use the syngas components CO, H2 and
CA
carbon and energy, or electron, source. Because of that, care must be taken to
prevent t.
in order to sustain the culture. However, should the feed gas supply be
interrupted, the
utilizing ethanol and C02 to produce acetic acid as seen in Equation (1)
below.
2 CH3-CH2-OH + 2 C02 4* 3 CH3-COOH Equation (1)
Through this reaction, the cells can advantageously gain electrons for
survival
from the oxidation of the alcohol to the carboxylic acid form. If feed gas is
interrupted,
the culture typically gains electrons, while not gaining carbon thus
decreasing cell
growth. It is therefore believed that this process provides a means of culture
survival,
though not optimized production. Cell washout, or the removal of any cells
from the
system, should be avoided in order to maintain the cell density during the
feed gas loss.
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This process leads to a buildup of acid. Steps must be taken to insure that
the
free acetic acid levels are maintained to concentration levels below
inhibiting
concentrations (<5g/L). This can be accomplished by raising the pH set point
to a range
of 5.1-5.3, increasing the liquid flow through the system thus lowering the
LRT to 15-20
hours, and by limiting the production of acid by limiting the available C02
and/or
ethanol or by lowering the culture metabolism through temperature reduction.
The production of acid depletes the ethanol concentration in the reactor.
Since
the culture is exhibiting decreased ethanol production while under these
conditions, the
ethanol concentration must be monitored to insure it is not excessively
depleted. Ethanol
may need to be added to the system or supplemented as the length of time
without feed
gas increases. During these experiments, ethanol concentration in the
bioreactor has
been depleted down to a concentration as low as 4g/L without detrimental
effects to the
culture.
Optionally, culture temperature plays vital role in this process as a way to
control
the metabolic rate of the cells. As the temperature is lowered, the cell's
metabolic rate
slows. That, in turn, slows the production of acid and the use of ethanol and
CO2.
Lowering the temperature when the reactor is without feed gas extends the
length of time
the culture can survive. Conversely, if the temperature is kept at 38 C the
acid
production rate is at its highest and careful monitoring of the acid level and
ethanol level
is required to keep the culture healthy. Experiments have lowered the culture
temperature to about 25 C, successfully maintaining the cell viability for
about 30 hours
without feed gas.
An embodiment of the present invention provides a delivery method of a
controlled amount of C02 comprising NaHCO3 addition. When sodium bicarbonate
is
introduced into an acid environment like the fermentation broth C02 is
produced as
shown below in Equation 2. It is believed that this method of C02 addition to
the
system is advantageous over sparging C02 into the culture because the sodium
bicarbonate not only adds C02 but also increases the culture pH to about 5.1,
helping to
compensate for the system production of acid by balancing the free acid
levels.
NaHCO3 + H+ p Na+ + H2CO3 p Na+ + H2O + C02(g) Equation 2
The conversion of ethanol to acid starts to take place almost immediately, or
within seconds, after the loss of the feed gas. It is possible to prevent a
quick and large
buildup of acid at the start of the feed gas loss by stripping the dissolved
C02 present in
the culture using a high N2 flow of about 400-450mL/min. Nitrogen should be
sparged
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through the culture for approximately 0-15 minutes as soon as possible after
the feed gas
flow is lost, the faster this is done the more advantageous it is for the
present invention.
Nitrogen flow within the first 5 minutes is an embodiment of the present
invention.
Once the inventory of dissolved C02 has been removed, the NaHCO3 addition can
be
started using a controlled feed rate.
Using NaHCO3 addition to provide C02 can increase the culture pH. If the cells
remain active utilizing all of the sodium bicarbonate available, the pH should
increase to
about 5.1 then remain there. This is a desired secondary effect and should not
be
prevented. The slow and steady increase in pH will help to counter the rising
acid
production by keeping the free acid level in check. If however, the cell
activity is
compromised, the pH will increase beyond about 5.3 giving an indication that
the culture
may be denatured or otherwise functionally decreased.
When the feed gas is available, the sodium bicarbonate addition should be
stopped and the mass transfer of the feed gas should be increased as quickly
as possible,
but taking care not to overwhelm the cells. Over a period of time of about 10-
15
minutes, the agitation should be increased back to the same setting used prior
to the feed
gas loss and the feed gas flow rate increased back to about 50% of the
original feed rate.
Because of the availability of substrate and the high level of total acetyl
the culture will
steadily convert the acid back into ethanol. This will be reflected in an
increase in pH
and is expected. Changes to the fermentor's feed gas flow rate during this
time should
be made based on gas conversions as in any normal reactor operations.
Once the feed gas flow has been restarted, if all has gone well in preserving
the
cell's viability, the feed gas flow rate should be able to reach a normal
operating setting
within about 20-26 hours. The ethanol and acid concentrations can take longer
to reach
normal operating levels, about 26-72 hours.
A minimum agitation is required to maintain the temperature distribution
throughout the bioreactor and to maintain the culture pH. Minimum agitation
would be
defined as just enough mixing to keep the liquid distributed. This can be
about 50rpm,
or 40-60rpm, as compared to high rates, such as 400rpm, used during normal
operations.
This agitation in effect also keeps the cells suspended. It is believed that
the cells should
be suspended in order to provide constant contact with CO2 and ethanol to
perform the
needed reactions.
Acetogenic microorganisms require CO or H2 and C02 in order to gain the
necessary electrons and carbon for cell growth. During the periods of time
without feed
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gas neither CO nor H2 are available for the cell growth process. It is
believed that cell
growth is suspended during those times of feed gas loss. It may be reasoned
that a lower
supply of feed gas could be used for culture survival during times in which
the feed gas
supply may be limited. Diminished amount of feed gas supplied provides the
culture
reversion to acid production mode. When the substrate feed rate is decreased,
the culture
will automatically stop the conversion of acid to ethanol causing an increased
drop in the
ethanol to acid ratio. Once this gas loss process is fully understood, it may
be advisable
that the best course of action is to stop the supply of feed gas completely
during times of
feed gas production difficulties rather that supplying a lower rate of
substrate. If it is
determined that it is preferable to lower the substrate feed rate, action must
be taken to.
cope with the increase in acid. Such actions would involve increasing the
liquid flow
through the system to remove acid, increasing the culture pH to maintain a
tolerable free
acid level, and/or removing a large portion of the cells from the system to
maintain a
healthy gas uptake to cell ratio for minimal acid production.
Table 1. Medium Component and Their Concentrations in the lx EtOH Medium
Component / Ion Added As 1 x EtOH
Conc in Med (ppm)
NH4+ NH4Cl / (NH4)2HPO4 838
Fe FeC12 = 41120 16.8
Ni NiC12 6H20 0.198
Co CoC12 = 61120 0.991
Se Na2Se03 0.0913
Zn ZnSO4 = 7H2O 0.455
Mo Na2MoO4 = 2H20 0.238
Mn MnC12 = 4H2O 0.167
B H3BO3 1.05
Cu CuC12 =21120 0.149
W Na2WO4 = 2H20 1.12
K KCl 78.6
Mg MgC12 = 6H20 59.8
Na NaCl 78.7*
Ca CaC12 = 2H20 54.5
Cysteine HCl Cysteine HCI 250
P04-2 H3PO4 / (NH4)2HPO4 816
Pantothenic Acid Pantothenic Acid 0.025
Biotin Biotin 0.020
Thiamin Thiamine 0.050
* Na+ concentration is from NaCl only. It does not include Na+ from the other
components such as Na2WO4 = 2H2O.
** Ca+2 concentration does not include calcium from pantothenic acid, calcium
salt.
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Table 2 details the culture parameters before and after the experiment such as
pH,
redox, ethanol and acetic acid. Generally, when the culture uses ethanol and
C02 for
survival, the ethanol level decreases as the acid concentration and culture pH
increases.
Table 2 also lists the high measured level of free acid during those
experiments as well
as the number of hours after the end of the experiment to recover to the
original gas feed
rate. It should be remembered that a key component in culture survival during
the feed
gas loss is the maintenance of a free acid concentration <5.0g/L. As acid is
being
produced, a higher culture pH and a faster acid removal rate is required to
prevent acid
inhibition. As acid is being produced, a higher culture pH or a faster acid
removal rate is
required to prevent acid inhibition.
Table 3 details the C02 addition to the culture in mmol/min per gram of cells
in
the culture. The calculations were based on the feed rate of sodium
bicarbonate and the
total number of cells in the bioreactor. At 25 C a C02 feed rate of
0.014mmol/min=g
was sufficient to sustain the culture for 12 hours without feed gas. When the
experimental length of time was increased to 24 hours, an average C02 feed
rate of
0.034mmol/min=g was required for healthy culture survival. Interestingly, when
the
culture temperature was increased to 38 C, the culture required a minimum C02
feed
rate of 0.114mmol/min=g to maintain a healthy culture. At 38 C the cell's
metabolism is
higher requiring more energy for survival, thus more ethanol conversion to
acid.
Example 2
Survival of the culture for 17 and 24 hours without feed gas
Experimental conditions:
16.9 hr without feed gas
Temperature decreased to 25 C
Medium addition was unchanged for the experiment
0.030mmol/min C02 feed rate per gram of cells
Permeate purge was used to hold in the cells
C02 was NOT stripped from the culture broth at the start of the experiment
Before the start of the experiment the culture cell density was 3.7g/L; pH was
4.44; redox was -440mV; CO and H2 uptake was 5.0 and 1.2mmol/min respectively;
CO
and H2 conversions were 86 and 40% respectively; ethanol was 23.5g/L; and acid
was
3.9g/L.
At t = 9511.6 hours, the feed gas flow rate was decreased from 474mL/min to
53mUmin. The agitation was dropped from about 400 to about 50rpm, and the
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temperature set point on the reactor was decreased from 38 to 25 C within
about 12
minutes. Once cooling was done, 38.5g/L sodium bicarbonate as started at
0.57mL/min
providing a 0.030mmol/min per gram of cells CO2 feed rate; the feed gas flow
was
stopped; a permeate purge was started at 1.95mL/min, and the medium flow was
kept at
1.37mL/min. Nitrogen was slowly added to the reactor headspace to prevent a
vacuum
from forming in the reactor. The culture was left in that condition for 16.9
hours.
During the experiment liquid samples were taken approximately every 2 hours to
monitor culture pH, cell density, products and cell morphology. The culture pH
increased steadily throughout the experiment to reach 5.07 toward the end of
the
experiment. The ethanol concentration decreased steadily from 23.5 to 7.0g/L
by the end
of the experiment. The total acetyl concentration increased steadily from 3.9
to 8.2g/L.
Approximately 12 hours into the experiment the culture morphology showed only
5-10%
of the cells were grainy or hollow bodies. The cell length was average with
mild to no
warping or bending.
At t = 9528.5hrs, the temperature set point on the reactor was increased back
to
about 38 C; the feed gas was restarted at about 53mL/min; Medium B and
permeate
purge were stopped; and the N2 flow into the reactor headspace was stopped.
When the
temperature reached about 28.0 C, the feed gas flow rate was increased to
143mL/min.
At about 30.0 C the feed gas flow was increased again to 236mL/min, or 50% or
the
original gas flow rate. At about 32.0 C, the agitation was increased to
200rpm. At about
34 C the agitation was increased to about 400rpm.
Initial conversions about 40 minutes after the increase in gas, agitation and
temperature were good at 47%H2 and 88%CO. Approximately 15min later the
conversions were still very good at 47%H2 and 87%CO. Gas flow rate increases
were
started right away. It took about 18.3 hours to reach the maximum gas flow
used prior to
the start of the experiment. As the gas flow rate was increased, the pH
continued to drop
reaching about 4.60 within about 18.3 hours. The ethanol increased back to
20.0g/L 40.6
hours after the end of the experiment, and the acid dropped back to 3.4g/L
after 24.9
hours.
Example 3
Experimental conditions:
About 24 hr without feed gas
Temperature dropped to about 25 C
Medium addition was unchanged for the experiment
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0.035mmol/min CO2 feed rate per gram of cells
Permeate purge was used to hold in the cells
CO2 was NOT stripped from the culture broth at the start of the experiment
Before the start of the experiment the culture cell density was about 3.2g/L;
pH
was about 4.50; redox was about -425mV; CO and H2 uptake was 4.7 and
1.5mmol/min
respectively; ethanol was about 17.7g/L; and acid was about 2.93g/L.
At t = 1888 hours, the feed gas flow rate was decreased from about 474mL/min
to 53mUmin. The agitation was dropped from about 400 to about 50rpm, and the
temperature set point on the reactor was decreased from about 38 to about 25 C
in about
14 minutes. Once cooling was done, the sodium bicarbonate addition was started
using a
about 38.5g/L NaHCO3 flow of 0.58mL/min providing a CO2 feed rate of
0.035mmol/min per gram of cells; the feed gas flow was stopped; a permeate
purge was
started at 1.8lmUmin, and the medium flow was kept at 1.30mUmin. Nitrogen was
slowly added to the reactor headspace to prevent a vacuum from forming in the
reactor.
The culture was left in that condition for about 24 hours.
Approximately 15.5 hours into the experiment the reactor condition provided:
cell density of about 2.4g/L; pH of about 4.96; EtOH of about 6.06g/L; and
acid was
about 7.87g/L. The cell morphology showed about 5-10% of the cells were grainy
or
almost grainy. Due to the low ethanol concentration left in the reactor, at t
= 1904 hours,
115mL of Gem Clear grain alcohol was added to 9L of medium A for an ethanol
concentration of about lOg/L. The medium feed rate remained the same providing
an
ethanol feed rate of 0.037mmol/min per gram of cells.
At the end of the 24 hours, the culture condition provided: cell density of
about
2.9g/L; pH of about 5.04; EtOH of about 4.10g/l; and acid was about 8.68g/L.
The cell
morphology showed about 10-15% of the cells had turned grainy or almost
grainy.
At t = 1912hrs, the temperature set point on the reactor was increased back to
about 38 C; the feed gas was restarted at about 53ml/min; Medium B and
permeate
purge were stopped; and the N2 flow into the reactor headspace was stopped.
Medium
was changed to a normal lx EtOH medium with no ethanol added. The feed gas and
agitation were increased at regular intervals as the temperature increased
stepwise.
When the temperature reached about 28.0 C, the feed gas flow rate was
increased to
about 179mL/min. At about 30.0 C the feed gas flow was increased again to
about
248ml/min, or about 50% or the original gas flow rate. At about 32.0 C, the
agitation
was increased to about 200rpm. At about 34 C the agitation was increased to
about
34
CA 02718132 2010-09-08
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400rpm.
As an embodiment, initial conversions about 35 minutes after the increase in
gas,
agitation and temperature were at 60%H2 and 84%CO. As an embodiment,
approximately 15min later the conversions provided: 62%H2 and 91 %CO. Gas flow
rate
increases were introduced immediately. In this case, it took about 19.5 hours
to reach
the maximum gas flow used prior to the start of the experiment.
Example 4
Experimental Conditions:
23.5 hr without feed gas
Temperature dropped to 25 C
Medium addition was reduced to half of the normal flow; the cysteine
concentration was doubled in the medium
0.039mmol/min C02 feed rate per gram of cells
Permeate purge was used to hold in the cells
C02 was NOT stripped from the culture broth at the start of the experiment
In an embodiment, the 24 hour gas loss experiment showed that the culture can
survive very well for about 24 hours without feed gas while providing
0.035mmol/min
C02 addition per gram of cells. The medium and sodium bicarbonate flows into
the
reactor during the experiment required about 2.6L of permeate to be removed to
prevent
cell loss due to washout. That is slightly more than the 2.4L of culture
volume. In lab
scale that ratio of required liquid flow to culture volume is well tolerated.
However, on
an industrial scale, the waste water amount must be monitored and, if needed,
decreased.
In this experiment all parameters were kept the same as the previous,
experiments except
the medium flow rate was reduced by half to reduce the amount of permeate
purge that is
required. There have been some indications in past experiment that suggest
that a
reduction in the cysteine feed rate may interfere with the experiment, so
during this
experiment the cysteine concentration in medium was doubled to retain the
cysteine feed
rate.
Before the start of the experiment the culture cell density was about 2.5g/L;
pH
was about 4.50; redox was about -440mV; CO and H2 uptake was about 4.8 and
about
1.2mmol/min respectively; ethanol was about 21.3g/L; and acid was about
2.96g/L.
At t = 2008.5 hours, the feed gas flow rate was decreased from 474mL/min to
53mLJmin. The agitation was dropped from about 400 to about 50rpm, and the
temperature set point on the reactor was decreased from 38 to 25 C within 13
minutes.
CA 02718132 2010-09-08
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Once cooling was done, the sodium bicarbonate addition was started using a
38.5g/L
NaHCO3 solution at 0.57ml/min; the feed gas flow was stopped; a permeate purge
was
started at 1.20ml/min, and the medium flow was reduced to 0.68m1/min. Nitrogen
was
slowly added to the reactor headspace to prevent a vacuum from forming in the
reactor.
The cysteine concentration was increased to 5g/L in medium A. The C02 was
provided
at 0.039mmol/min per gram of cells. The culture was maintained in that
condition for 24
hours.
During the experiment liquid samples were taken approximately every 2 hours to
monitor culture pH, cell density, products and cell morphology. As expected
the pH
increased steadily throughout the experiment to reach 5.14 at the end of the
experiment.
The ethanol concentration decreased steadily from 21.3 to 6.03g/L by the end
of the
experiment. The total acetyl concentration increased steadily from 2.96 to
10.38g/L.
After about 24 hours the culture morphology showed about 10-20% of cells were
grainy
or almost grainy.
At t = 2032hrs, the temperature set point on the reactor was increased back to
about 38 C; the feed gas was restarted at about 53ml/min; sodium bicarbonate
addition
and permeate purge were stopped; and the N2 flow into the reactor headspace
was
stopped. Medium flow rate was increased back to 1.37ml/min. As the culture was
heating the feed gas flow was increased back to 248ml/min and the agitation
was raised
to about 400rpm stepwise.
Initial conversions about 30 minutes after the increase in gas, agitation and
temperature were at 50%H2 and 87%CO. Gas flow rate increases were started
right
away. It took about 24 hours to reach the maximum gas flow used prior to the
start of
the experiment.
Example 5
Minimization of the CO2 feed rate while at 25 C, 12 hour culture survival
Experimental Conditions:
12 hr without feed gas
Temperature dropped to 25 C
Medium addition was unchanged for the experiment
0.014mmol/min C02 feed rate per gram of cells
Permeate purge was used to hold in the cells
C02 was NOT stripped from the culture broth at the start of the experiment
This experiment evaluates the minimum C02 addition rate needed to sustain the
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culture for 12 hours at 25 . As an embodiment, NaHCO3 solution used as Medium
B
was decreased in concentration while keeping all other experimental parameters
the
same. In this experiment the NaHCO3 concentration was dropped to about 19.3g/L
providing a C02 feed rate of about 0.014mmol/min C02 per gram of cells in the
reactor.
This is a low feed rate with culture survival.
Before the start of the experiment the culture cell density was about 4.0g/L;
pH
was about 4.43; redox was about -430mV; CO and H2 uptake was about 4.9 and
about
1.3mmol/min respectively; CO and H2 conversions were about 86 and about 44%
respectively; ethanol was about 18.9g/L; and acid was about 3.8g/L.
At 2015, t = 9580.7 hours, the feed gas flow rate was decreased from about
474mL/min to about 53mL/min. The agitation was dropped from about 400 to about
50rpm, and the temperature set point on the reactor was decreased from about
38 to
about 25 C within about 12 minutes. Once cooling is accomplished, the sodium
bicarbonate flow was started at 0.56mJJmin; the feed gas flow was stopped; a
permeate
purge was started at 1.96mL/min, and the medium flow was kept at 1.36mL/min.
Nitrogen was slowly added to the reactor headspace to prevent a vacuum from
forming
in the reactor. The culture was maintained in that condition for about 12
hours.
During the experiment liquid samples were taken approximately every 2 hours to
monitor culture pH, cell density, products and cell morphology. Throughout the
experiment the pH increased steadily throughout the experiment to reach about
4.72
toward the end of the experiment. The ethanol concentration decreased steadily
from
about 18.9 to about 10.7g/L by the end of the experiment. The total acetyl
concentration
increased steadily from about 3.8 to about 6.0g/L. The cell density dropped
from about
4.0 to about 2.8g/L. After about 12 hours the culture morphology provided
about 95+%
of the cells were average to slightly long in length with only minimal warping
or bending
and only an occasional grainy cell or hollow body.
At t = 9592.7hrs, the temperature set point on the reactor was increased back
to
about 38 C; the feed gas was restarted at about 53mL/min; Medium B and
permeate
purge were stopped; and the N2 flow into the reactor headspace was stopped.
While the
culture warmed over the next 15 minutes, the feed gas flow rate and agitation
were
increased stepwise. When the temperature reached about 28.0 C, the feed gas
flow rate
was increased to about 178mL/min. The pH of the culture was slowing dropping
indicating culture activity. At about 30.0 C the feed gas flow was increased
again to
236mL/min, or 50% or the original gas flow rate. At about 32.0 C, the
agitation was
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increased to about 200rpm. At about 34 C the agitation was increased to about
400rpm.
Initial conversions 50 minutes after the increase in gas, agitation and
temperature
provided about 53%H2 and about 89%CO. Gas flow rate increases were started
immediately. It took 14.6 hours to reach the maximum gas flow used prior to
the start of
the experiment. As the gas flow rate was increased, the ethanol concentration
increased
back to about 23.7g/L 49.4 hours, and the acid dropped back to about 3.5g/L
9.3 hours
after experiment end.
Example 6
Minimization of the C02 feed rate while at 38 C, 6 hour culture survival
Experiment Conditions:
6 hr without feed gas
Temperature remained at 38 C
Medium addition was unchanged for the experiment
0. 1 14mmol/min C02 feed rate per gram of cells
Permeate purge was used to hold in the cells
N2 used to strip C02 from the culture broth at the start of the experiment
Before the start of the experiment the culture cell density was about 2.76g/L;
pH
was about 4.60; redox was about -440mV; CO and H2 uptake was about 4.5 and
about
l.2mmol/min respectively; ethanol was about 19.0g/L; and acid was about
2.43g/L.
At t = 3080.5hrs, the feed gas flow rate was decreased from about 475ml/min to
about 53m1/min then turned off. A high N2 flow was started through the feed
gas
sparger while the agitation was still at about 400rpm for about 3 minutes to
strip the CO2
from the culture. While stripping the C02, the pH control was turned off to
prevent any
sodium bicarbonate addition. After about 3 minutes, the N2 flow was dropped
and the
N2 inlet was changed to the headspace rather than the sparger. The agitation
was
dropped from about 400 to about 50rpm. C02 addition was started at about
0.82mL/min
using about 77g/L NaHCO3 to provide about 0. 1 14mmol/min C02 per gram of
cells in
the reactor. The medium flow rate was left at about 1.34mL/min, and a permeate
purge
flow of 2.25ml/min was started.
The experiment was stopped after 6 hours at t = 3086.5 hours due to high acid.
Sodium bicarbonate addition, permeate purge and N2 addition to the headspace
were
stopped. The feed gas flow was restarted at 53mL/min. The feed gas and
agitation were
increased at about the same intervals as is used to increase gas and agitation
when the
culture is warming to about 38 C in the previous experiments. Two minutes
after the
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experiment was stopped the feed gas flow was increased to about 170mL/min.
About
four minutes after the experiment was stopped the feed gas flow was increased
to about
248mL1min (50% of the original gas flow rate). About six minutes after the
experiment
was stopped the agitation was increased to 200rpm. About eight minutes after
the
experiment was stopped the agitation was increased to about 400rpm.
After about 6 hours with C02 addition and low agitation, the pH was about
5.08.
A liquid analysis showed the products were about 10.4g/L ethanol and about
8.21 g/L
acid. The culture morphology showed about 3% of the cells were grainy with an
additional about 22% were almost grainy.
Initial conversions about 30 minutes after the experiment was stopped were
about
47% H2 and about 87% CO. Gas flow rate increases were started right away. The
original feed gas flow rate was reached at t = 3108 hours, or about 21.5 hours
after the
experiment ended.
Table 2. Culture Parameters in Gas Loss Experiments at about 25C and about 38C
Where Culture was restarted using Agitation and at Least 50% of the Original
GFR
xperlHrs Max Recovery
Emental Bicarbonate W/o A in EtOH A in Hac Ain pH A in Redox Free Time
End End End End
Culturue Feed Before of Before of Before of Before of Acid # Hrs to
Run Temp Conc Gas Exp Exp Exp Exp Exp Exp Exp Exp Reached Reach
Original
(C) mV GFR
1 25 77 8 21.01 10.74 4.41 9.36 4.48 4.93 -425 325 4.1 29.2
2 25 38.5 16.9 23.51 7.03 3.91 8.24 4.44 5.07 -440 245 3.5 18.3
3 25 19.25 12 18.95 9.91 3.83 5.78 4.43 4.72 -430 265 3.2 14.6
4 25 9.625 12.25 20.28 11.92 3.54 2.61 4.53 5.12 -440 240 2.3 35.2
5 25 14.63 12.25 17.89 11.10 5.52 4.40 4.53 5.04 -440 370 1.5 35.6
6 25 38.5 24 17.70 4.10 2.93 8.68 4.50 5.04 -425 305 3.1 19.5
7 25 38.5 23.5 21.30 6.03 2.96 10.38 4.50 5.14 -440 320 3.5 24
8 38 77 4 21.30 9.88 3.28 14.60 4.56 5.26 -445 340 3.4 13.5
9 38 77 6.75 23.20 7.25 3.15 14.50 4.47 5.53 -430 280 3.2 19
10 38 77 6.5 20.44 7.04 2.86 12.79 4.53 5.30 -440 235 3.1 20.75
11 38 77 7 22.20 8.77 3.40 14.10 4.48 5.15 -435 275 4.0 28
12 38 77 6 19.00 10.4 2.43 8.21 4.60 5.08 -440 270 2.6 21.5
13 38 77 8.5 20.15 7.5 1.75 12.12 4.53 5.2 -430 200 3.4 30.5
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Table 3. Calculated C02 Feed Rate per Gram of Cells in the Reactor (mmol/min
g) for
Bicarbonate Addition, Feed Gas Loss Experiments w/Known C02 Addition Rates
/
Run Culture NaHCO3 NaHCO3 C02 Feed Cell Culture C02 Feed Rate Exp Time
Recovery
per Gram of
Temp Conc Flow Rate Rate Density Vol Cells Length Time
(mmol/min
(C) (g/ L) mmol/min (L) (Firs) (Hrs)
1 25 77 0.57 0.5224 3.64 2.35 0.0610 8 29.2
2 25 38.5 0.57 0.2612 3.71 2.325 0.0303 17 18.3
3 25 19.25 0.56 0.1283 4.02 2.325 0.0137 12 14.6
4 25 9.625 0.57 0.0653 3.25 2.3 0.0087 12 35.2
25 14.63 0.58 0.1010 3.97 2.375 0.0107 12 35.6
6 25 38.5 0.58 0.2658 3.20 2.4 0.0347 24 19.5
7 25 38.5 0.57 0.2612 2.69 2.5 0.0388 24 24
8 38 77 3.18 2.9147 3.12 2.45 0.3815 4 13.5
9 38 77 1.65 1.5123 2.48 2.45 0.2492 6.75 19
38 77 1.4 1.2832 2.98 2.35 0.1834 6.5 20.75
11 38 77 1 0.9166 2.84 2.4 0.1347 7 28
12 38 77 0.82 0.7516 2.76 2.4 0.1135 6 21.5
13 38 77 0.92 0.8432 2.71 2.4 0.1298 8.5 30.5
Table 4. C02 Feed Rate, EtOH Uptake Rate and Acid Production Rate During the
Feed
Gas Loss Experiments
Run Experiment C02 Acid EtOH Experiment
Temperature Feed Rate Production Consumption Length of Time
hrs
C mmol/min g) mmoilmin g) mmol/min g)
1 25 0.0610 0.0816 0.0442 8
2 25 0.0306 0.0419 0.0310 17
3 25 0.0140 0.0263 0.0211 12
4 25 0.0346 0.0451 - 24.0
5 25 0.0387 0.0546 0.0504 24.0
6 38 0.382 0.285 0.096 4.0
7 38 0.223 0.295 0.222 6.75
8 38 0.187 0.192 0.131 6.5
9 38 0.142 0.196 0.123 7.0
10 38 0.113 0.136 0.086 6.0
11 38 0.130 0.166 0.100 8.5
CA 02718132 2010-09-08
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Example 7
Comparative Example: An Exemplary Method of the Present Invention
A synthesis or waste gas containing CO and/or carbon dioxide/gaseous hydrogen
is continuously introduced into a stirred tank bioreactor containing a strain
of C.
ljungdahlii, along with a conventional liquid medium containing vitamins,
trace metals
and salts.
During method start-up using a culture inoculum of 10% or less the reactor is
operated with a batch liquid phase, where the liquid medium is not fed
continuously to
the reactor. The liquid phase in the reactor thus consists of a batch of
nutrient medium
with a nominal concentration of limiting nutrient, either calcium pantothenate
or cobalt.
Alternatively, a rich medium containing yeast extract, trypticase or other
complex
nutrients can also be employed.
Ideally, the gas phase at start-up is C02 free and contains excess H2.
The
gas rate and agitation rate are kept at low levels (less than 500 rpm in a New
Brunswick
Scientific Bioflo® fermentation bioreactor) to yield CO and H2 in
slight
excess, but at the same time, avoiding CO substrate inhibition. In a one-liter
laboratory
New Brunswick Scientific Bioflo® fermentation bioreactor, as an example,
where
the feed gas composition is 63% H2, 32% CO and 5% CH4, the agitation
rate
to initiate start-up is 400 rpm and the gas rate is 20 ml/min. The cause of
ethanol
production during start-up is excess H2; limitation on nutrients occurs
later. Thus,
excess liquid nutrients (pantothenate, cobalt) are actually present during
start-up to avoid
unwanted culture acclimation to low nutrients.
As the fermentation proceeds over a period of several hours after inoculation,
C02 is produced from the conversion of CO, and H2 is consumed along with
the
CO2, which is a signal to nominally increase the agitation rate to avoid
gas mass
transfer limitation. In the New Brunswick Scientific Bioflo® CSTR, the
exit gas is
25% CO, 67% H2, 2% CO2, and 6% CH4. If the agitation rate is
increased
too fast, CO substrate inhibition occurs, as evidenced by a decrease in
methane
concentration after an increase in agitation. Thus the agitation rate might
typically be
increased by 200 rpm in 24 hours. This procedure of monitoring CO2
production (or
H2 conversion) while nominally increasing agitation rate occurs at a
relatively rapid
rate until the target agitation rate is reached. A typical target agitation
rate in the New
Brunswick Scientific Bioflo® fermentation bioreactor is 900 rpm. During
this time
of increasing agitation rate in batch liquid culture, cell production instead
of product
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formation is of utmost importance. Thus, cell concentrations of about 1.5 g/L
are
attained, while typical product concentrations are 10 g/L ethanol and 2 g/L
acetate from
the batch culture.
Once the target agitation rate is reached, the system is allowed to grow to
maximum H2 uptake. It is desirable to have very high H2 exit
concentrations
(typically >60%) to assure ethanol production while limiting acetic acid
production. The
liquid medium feed is then turned on (for systems having batch inoculation
from stock
culture) to initiate continuous liquid feed and the gas feed rate is increased
toward the
target flow rate. In the laboratory New Brunswick Scientific Bioflo®
fermentation
bioreactor the liquid feed rate is typically 0.5 mL/min, while the gas flow
rate is
increased by 10 to 15% every 24 hours toward a target rate of 125 mL/min.
It is important to provide excess H2 in the feed gas to avoid excess
acetic
acid production. As the gas flow rate is increased, cell production increases
until the
reactor is eventually limited on liquid phase nutrients (calcium pantothenate
or cobalt) as
evidenced by a small drop in H2 conversion, at the target productivity.
In the New
Brunswick Scientific Bioflo® CSTR, this is recognized by a 10% drop in
H2
conversion at a target productivity of 20 g/Lday.
The production method and reactor system are then maintained at a steady state
producing 15 to 35 g/L ethanol and 0 to 5 g/L acetate as products, with only
occasional
small adjustments in limiting nutrients, liquid rates and gas rate. Typical
steady state
conditions in the laboratory New Brunswick Scientific Bioflo® fermentation
bioreactor without cell recycle, are a gas retention time (gas flow
rate/reactor liquid
volume) of 20 minutes, a liquid retention time (liquid flow rate/reactor
liquid volume) of
hours and an agitation rate of 900 rpm, yielding CO conversions of 92% and
H2
25 conversions of 60% with pantothenate limitation.
In an embodiment of this method in which cell recycle is added to the reactor
system, it is added at this time along with an adjustment in gas rate
(increase) and
nutrient concentration (decrease). With cell recycle in the New Brunswick
Scientific
Bioflo® CSTR, the gas retention time is typically 8 minutes, the liquid
retention
30 time is 12 hours, the cell retention time is 40 hours and the agitation
rate is 900 rpm.
These conditions typically yield a CO conversion of 92% and a H2
conversion of
50% with pantothenate limitation.
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Example 8
Comparative Example: Recovery from Severe Method Upset
A CSTR with cell recycle containing C. ljungdahlii, strain C-01 being
continuously fed gas and liquid nutrients and producing 15-35 g/L ethanol and
0-5 g/L
acetate at a steady state is upset due to unforeseen changes in method
conditions, e.g.,
mechanical problems in the reactor. Upset to the reactor system can either be
minor, such
as a brief increase in the gas rate which causes short-term substrate
inhibition, or major,
such as a longer term increase in the gas rate which eventually leads to
increased acetic
acid production and more severe molecular acetic acid product inhibition.
Short-term upsets are easily corrected by merely readjusting the upset
parameter
(for example, lowering the gas rate to its original level) and monitoring the
progress of
the reactor to assure that the upset has not led to a longer-term problem.
However, acetic acid product inhibition is a more severe problem. If excess
molecular acetic acid is produced by the culture as a result of long term
substrate
inhibition, excess nutrient addition, CO2 accumulation or mechanical
problems of
many types, the problem that led to the excess acetic acid must first be
corrected. The
excess acetic acid, which quickly leads to product inhibition, is then cleared
from the
system by increasing the liquid rate to wash the acetic acid (and
unfortunately ethanol)
from the system. Once the acetate level is below 3-5 g/L, the liquid rate is
reset and the
reactor is placed back under either excess H2 feed, or vitamin or cobalt
limitation
(with or without cell recycle). Bringing the reactor back involves reducing
the gas rate to
avoid substrate inhibition and/or agitation rate before cell washout and lysis
takes place.
The agitation rate or gas rate is then increased.
In one specific example, a CSTR with cell recycle containing C. ljungdahlii,
strain C-O1 that was producing ethanol and acetic acid from CO, CO2 and
H2
began producing acetic acid in response to a mechanical problem. The 2100 ml
reactor
was fed gas containing 62% H2, 31% CO and 7% C2H6 at a gas
retention
time of 15 minutes, and was operating with an agitation rate of 600 rpm and a
pH of
4.86. The liquid retention time was 23 hours and the cell retention time was
68 hours. B-
vitamin solution (an aqueous mixture of 50.5 mg/l calcium pantothenate, 20.6
mg/L d-
biotin and 50.6 mg/L thiamine HCl) was present in the liquid nutrient medium
containing salts and vitamins at a concentration of 0.4 ml vitamin solution
per liter of
medium (see Table 2). The ethanol concentration fell to 7 g/L, while the
acetate
concentration rose to 7 g/L, conditions that are neither stable for operating
the reactor nor
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CA 02718132 2010-09-08
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economical for ethanol production. The cell concentration was 2.4 g/L, the CO
conversion was 85% and the H2 conversion was 25%.
The strategy used in recovering the reactor consisted of first dramatically
reducing the gas feed rate to the reactor, followed by gradual recovery of the
reactor in
the presence of excess H2. The liquid rate to the reactor was not reduced
to clear
product inhibition in this example because the acetate concentration was not
exceedingly
high. Instead, the acetate concentration was allowed to more gradually drop to
non-
inhibiting levels with the reduction in gas flow rate and subsequent operation
in the
presence of excess H2. The specific procedure in recovering the reactor
is discussed
below.
Cell recycle was turned off and the gas rate was dramatically reduced by 70%
to
a gas retention time of 62 minutes, while only slightly adjusting the liquid
retention time
from 23 to 30 hours (t=0). The vitamin concentration in the medium was not
changed.
With this change in gas rate the CO conversion increased to 98% and the
H2
conversion increased to 80%. More importantly the system had excess H2
present,
as evidenced by the decrease in CO2 in the outlet gas from 19 to 5%. With
the onset
of excess H2, the acetate concentration fell while the ethanol
concentration
increased. At t=66 hr (66 hr after turning off cell recycle), for example, the
acetate
concentration had fallen to 4 g/L and the ethanol concentration had risen
slightly to 7.5
g/L.
The presence of excess H2 (and the lowered acetate concentration)
permitted
subsequent increases in as rate, first slowly and then at a faster rate. By
t=215 hr the gas
retention was 29 minutes, the ethanol concentration was 12 g/L and the acetate
concentration was 3 g/L. The ethanol productivity was 8 g/Lday. CO2 was
present
in the outlet gas at 6%, the CO conversion was 98% and the H2 conversion
was
80%. By t=315 hr, the ethanol concentration was 16 g/L and the acetate
concentration
was 4 g/L, again with good gas conversions, and a gas retention time of 20
minutes. The
ethanol productivity was 11 g/Lday. By t--465 hr, the ethanol concentration
had reached
20 g/L, with 3.5-4 g/L acetate also present. The ethanol productivity was 16
g/Lday. The
gas retention time had been dropped to 16 minutes, with CO and H2
conversions of
95 and 73%, respectively. These conditions were maintained for nearly 200
hours of
continuous operation, demonstrating that the reactor system had recovered its
ability to
produce ethanol and had essentially retained the previous operating
conditions.
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All published documents are incorporated by reference herein. Numerous
modifications and variations of the present invention are included in the
above-identified
specification and are expected to be obvious to one of skill in the art. Such
modifications
and alterations to the compositions and methods of the present invention are
believed to
be encompassed in the scope of the claims appended hereto.
