U.S. patent application number 13/812886 was filed with the patent office on 2013-05-23 for fermentation process for the production of organic acids.
This patent application is currently assigned to Myriant Corporation. The applicant listed for this patent is Theron Hermann, James Reinhardt, Lauren Staples, Russell Udani, Xiaohui Yu. Invention is credited to Theron Hermann, James Reinhardt, Lauren Staples, Russell Udani, Xiaohui Yu.
Application Number | 20130130339 13/812886 |
Document ID | / |
Family ID | 45560003 |
Filed Date | 2013-05-23 |
United States Patent
Application |
20130130339 |
Kind Code |
A1 |
Hermann; Theron ; et
al. |
May 23, 2013 |
FERMENTATION PROCESS FOR THE PRODUCTION OF ORGANIC ACIDS
Abstract
This invention relates to improvements in the fermentation
process used in the production of organic acids from biological
feedstock using bacterial catalysts. The improvements in the
fermentation process involve providing a fermentation medium
comprising an appropriate form of inorganic carbon, an appropriate
amount of aeration and a biocatalyst with an enhanced ability to
uptake and assimilate the inorganic carbon into the organic acids.
This invention also provides, as a part of an integrated
fermentation facility, a novel process for producing a solid source
of inorganic carbon by sequestering carbon released from the
fermentation in an alkali solution.
Inventors: |
Hermann; Theron; (Arlington,
MA) ; Reinhardt; James; (Columbus, OH) ; Yu;
Xiaohui; (Woburn, MA) ; Udani; Russell;
(Somerville, MA) ; Staples; Lauren; (Wilmington,
MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Hermann; Theron
Reinhardt; James
Yu; Xiaohui
Udani; Russell
Staples; Lauren |
Arlington
Columbus
Woburn
Somerville
Wilmington |
MA
OH
MA
MA
MA |
US
US
US
US
US |
|
|
Assignee: |
Myriant Corporation
Quincy
MA
|
Family ID: |
45560003 |
Appl. No.: |
13/812886 |
Filed: |
July 30, 2011 |
PCT Filed: |
July 30, 2011 |
PCT NO: |
PCT/US11/46047 |
371 Date: |
January 29, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61400596 |
Jul 31, 2010 |
|
|
|
Current U.S.
Class: |
435/145 |
Current CPC
Class: |
C12P 7/46 20130101 |
Class at
Publication: |
435/145 |
International
Class: |
C12P 7/46 20060101
C12P007/46 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] The invention was made with United States government support
under a contract awarded from the US Department of Energy under
Award Number DE-EE0002878/001. The United States government has
certain rights in the invention.
Claims
1. (canceled)
2. (canceled)
3. (canceled)
4. (canceled)
5. (canceled)
6. (canceled)
7. (canceled)
8. (canceled)
9. (canceled)
10. (canceled)
11. (canceled)
12. (canceled)
13. (canceled)
14. (canceled)
15. (canceled)
16. (canceled)
17. A method for producing succinic acid through fermentation
comprising the steps of: providing a biocatalyst for succinic acid
production; providing a source of organic carbon; providing a
source of neutralizing agent; providing a source of inorganic
carbon; and maintaining the biocatalysts under microaerobic
condition during the production phase.
18. The method for producing succinic acid according to claim 17,
wherein said source of neutralizing agent is ammonium
hydroxide.
19. The method for producing succinic acid according to claim 17,
wherein said source of inorganic carbon is ammonium
bicarbonate.
20. The method for producing succinic acid according to claim 17,
wherein said source of neutralizing agent is ammonium hydroxide and
said source of inorganic carbon is ammonium bicarbonate.
21. The method for producing succinic acid according to claim 17,
wherein the biocatalyst has an enhanced ability for inorganic
carbon uptake.
22. The method for producing succinic acid according to claim 21,
wherein the enhanced ability for inorganic carbon uptake results
from a genetic modification that increases inorganic bicarbonate
transport activity.
23. The method for producing succinic acid according to claim 17,
wherein the biocatalyst has an enhanced ability for inorganic
carbon assimilation.
24. The method for producing succinic acid according to claim 23,
wherein the enhanced ability for inorganic carbon assimilation of
the biocatalyst results from a genetic modification to one of the
carboxylating enzyme present within the biocatalyst.
25. The method for producing succinic acid according to claim 17,
wherein the fermentation is run in a batch mode.
26. The method for producing succinic acid according to claim 17,
wherein the fermentation is run in a fed-batch mode.
27. The method for producing succinic acid according to claim 17,
wherein the biocatalyst for succinic acid production is obtained
from group consisting of Escherichia coli, Gluconobacter oxydans,
Gluconobacter asaii, Achromobacter delmarvae, Achromobacter
viscosus, Achromobacter lacticum, Agrobacterium tumefaciens,
Agrobacterium radiobacter, Alcaligenes faecalis, Arthrobacter
citreus, Arthrobacter tumescens, Arthrobacter paraffineus,
Arthrobacter hydrocarboglutamicus, Arthrobacter oxydans,
Aureobacterium saperdae, Azotobacter indicus, Brevibacterium
ammoniagenes, divaricatum, Brevibacterium lactofermentum,
Brevibacterium flavum, Brevibacterium globosum, Brevibacterium
fuscum, Brevibacterium ketoglutamicum, Brevibacterium helcolum,
Brevibacterium pusillum, Brevibacterium testaceum, Brevibacterium
roseum, Brevibacterium immariophilium, Brevibacterium linens,
Brevibacterium protopharmiae, Corynebacterium acetophilum,
Corynebacterium glutamicum, Corynebacterium callunae,
Corynebacterium acetoacidophilum, Corynebacterium acetoglutamicum,
Enterobacter aerogenes, Erwinia amylovora, Erwinia carotovora,
Erwinia herbicola, Erwinia chrysanthemi, Flavobacterium peregrinum,
Flavobacterium fucatum, Flavobacterium aurantinum, Flavobacterium
rhenanum, Flavobacterium sewanense, Flavobacterium breve,
Flavobacterium meningosepticum, Micrococcus sp. CCM825, Morganella
morganii, Nocardia opaca, Nocardia rugosa, Planococcus eucinatus,
Proteus rettgeri, Propionibacterium shermanii, Pseudomonas
synxantha, Pseudomonas azotoformans, Pseudomonas fluorescens,
Pseudomonas ovalis, Pseudomonas stutzeri, Pseudomonas acidovolans,
Pseudomonas mucidolens, Pseudomonas testosteroni, Pseudomonas
aeruginosa, Rhodococcus erythropolis, Rhodococcus rhodochrous,
Rhodococcus sp. ATCC 15592, Rhodococcus sp. ATCC 19070,
Sporosarcina ureae, Staphylococcus aureus, Vibrio metschnikovii,
Vibrio tyrogenes, Actinomadura madurae, Actinomyces
violaceochromogenes, Kitasatosporia parulosa, Streptomyces
coelicolor, Streptomyces flavelus, Streptomyces griseolus,
Streptomyces lividans, Streptomyces olivaceus, Streptomyces
tanashiensis, Streptomyces virginiae, Streptomyces antibioticus,
Streptomyces cacaoi, Streptomyces lavendulae, Streptomyces
viridochromogenes, Aeromonas salmonicida, Bacillus pumilus,
Bacillus circulans, Bacillus thiaminolyticus, Escherichia freundii,
Microbacterium ammoniaphilum, Serratia marcescens, Salmonella
typhimurium, Salmonella schottmulleri, Bacillus subtilis, Bacillus
licheniformis, Bacillus amylolliquefaciens and Xanthomonas
citri.
28. The method of producing succinic acid as in claim 17, wherein
the organic carbon source is derived from the hydrolysis of a plant
derived carbohydrate.
29. The method of producing succinic acid as in claim 17, wherein
the organic carbon is derived from the hydrolysis of starch from
grain sorghum.
30. The method of producing succinic acid as in claim 17, wherein
the organic carbon is derived from the hydrolysis of
lignocellulosic feed stock.
31. The method for producing succinic acid as in claim 20, wherein
the ammonium hydroxide and ammonium bicarbonate solution are mixed
together in advance and provided to the fermentor through a single
feed line
32. The method for producing succinic acid as in claim 20, wherein
the ammonium hydroxide and ammonium bicarbonate are used in the
molar ratio of 8:1 to 1:1.
33. The method for producing succinic acid as in claim 20, wherein
the ammonium hydroxide and ammonium bicarbonate are used in the
molar ratio of 4:1 to 2:1.
34. The method for producing succinic acid as in claim 20, wherein
the ammonium bicarbonate is prepared by trapping carbon dioxide gas
in ammonium hydroxide solution.
35. The method of producing succinic acid as in claim 17, wherein
the microaeration is provided by mixing air to carbon dioxide gas
in the amount of less than 1 percent of air and feeding the gas
mixture at the flow rate of at least 0.001 vvm.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the priority of the U.S. Provisional
Application Ser. No. 61/400,596, filed on Jul. 31, 2010.
BACKGROUND OF THE INVENTION
[0003] A 2004 U.S. Department of Energy report entitled "Top value
added chemicals from biomass" has identified twelve building block
chemicals that can be produced from renewable feedstocks. The
twelve sugar-based building block chemicals are 1,4-diacids
(succinic, fumaric and maleic), 2,5-furan dicarboxylic acid,
3-hydroxy propionic acid, aspartic acid, glucaric acid, glutamic
acid, itaconic acid, levulinic acid, 3-hydroxybutyrolactone,
glycerol, sorbitol, and xylitol/arabinitol.
[0004] Building block chemicals are molecules with multiple
functional groups that possess the potential to be transformed into
new families of useful molecules. The twelve building blocks
identified by U.S. Department of Energy can be subsequently
converted to a number of high-value bio-based chemicals or
materials.
[0005] During the last few years a number of microorganisms have
been created through genetic engineering for the production of
industrially useful monomeric building block chemical compounds.
Many natural metabolites derived from biological fermentative
processes such as dicarboxylic acids, amino acids, and diols have
functional groups that are suitable for polymerization and chemical
synthesis of industrially useful polymers.
[0006] In recent years attention has been focused on reducing the
cost of production of industrially useful chemical compounds
through biological fermentation. One well known approach for
reducing the cost of fermentative production of chemical compounds
is to use low-cost minimal medium in place of expensive
nutritionally rich medium. For example, the E. coli strain
described in U.S. Pat. No. 7,223,567 requires a rich medium
supplemented with glucose as the source of carbon for the
production of succinic acid. The E. coli strain KJ122 useful for
the production of succinic acid described by Jantama et al (2008a
and 2008b) and in the PCT Patent Application Publications Nos.
WO/2008/021141A2 and WO/2010/115067 is capable of growth on a
minimal medium without the need for any expensive ingredients such
as yeast extract or tryptone. Another approach that is being
attempted to further reduce the cost of fermentative production of
chemical compounds is to replace the currently used expensive
feedstocks such as dextrose and sucrose with cheaper organic carbon
source such as a mixture of six-carbon and five-carbon sugars
derived form lignocellulosic biomass through a pretreatment
process.
[0007] The inventors have discovered a novel method for further
reducing the cost of producing specialty chemicals through
biological fermentation. This new method for improving the
productivity and the yield of succinic acid through a biological
fermentation process is based on the observation that the yield and
productivity of succinic acid in the biological fermentation
process requires the supply of both organic carbon and inorganic
carbon sources. A reduction in the cost of production of succinic
acid can be achieved by means of supplying the required inorganic
carbon in a cost effective manner besides meeting the requirement
for organic carbon sources.
[0008] As defined in this invention, the term organic carbon refers
to the organic feedstocks such as xylose, glucose, glycerol and
sucrose useful for the fermentative production of organic acid by
the microorganism. The term inorganic carbon refers to the carbon
dioxide present in the gas phase of the fermentation chamber and
the carbonate and bicarbonate salts added as a component of the
fermentation medium.
[0009] The importance of the contribution from inorganic carbon
towards succinic acid production by microbial catalysts is now well
established although the relative contribution of inorganic and
organic carbon fractions to the final succinic acid production is
not precisely established.
[0010] While the transformation of organic carbon into succinic
acid is achieved by the modification of the central metabolic
pathway including the glycolytic pathway and the tricarboxylic acid
cycle within the cell, the incorporation of inorganic carbon into
succinic acid requires the participation of carboxylating enzymes.
At least four different types of carboxylating enzymes are known to
be functional within bacterial cells. The phosphoenol pyruvate
carboxylase (PEBcase or PPC) carboxylates phosphoenol pyruvate
leading to the formation of oxaloacetic acid. The malic enzyme
carboxylates pyruvic acid leading to the formation of malic acid
and requires reduced cofactors such as NADH or NADPH. The third
carboxylating enzymes known as pyruvate carboxlase (PYC)
carboxylates pyruvic acid to produce oxaloacetic acid. The fourth
carboxylating enzyme known as phosphoenolpyruvate carboxykinase
(PCK) carboxylates phosphoenol pyruvate to oxaloacetate with the
production of one molecule of ATP for every molecule of
oxaloacetate produced from the carboxylation of a phosphoenol
pyruvate molecule. The inorganic carbon assimilated through the
carboxylation reactions mediated by one of these four different
carboxylating enzymes present within a bacterial cell contributes
to the carbon back bone of the succinic acid produced through
fermentation process.
[0011] The E. coli strains currently in use for the production of
succinic acid are reported to have enhanced activity for one or
other carboxylating enzymes. U.S. Pat. No. 6,455,284 discloses the
use of an exogenous pyruvate carboxylase enzyme for enhancing the
production of oxaloacetate-derived chemicals through fermentation.
Expression of Rhizobium etli pyruvate carboxylase gene in E. coli
cells caused an increased carbon flow towards oxaloacetate in wild
type E. coli cells without affecting the glucose uptake rate or the
growth rate and restored succinate formation in E. coli
phosphoenolpyruvate carboxylase null mutants. Zhang et al (2009)
have reported that in KJ122 strain of E. coli due to a mutation in
the promoter region, the phosphoenolpyruvate carboxykinase enzyme
shows enhanced carboxylation capacity.
[0012] Sanchez et al (2005) have reported that the flux to the
oxaloacetate pool was increased by overexpressing the enzyme
pyruvate carboxylase (PYC) from Lactococcus lactis in E. coli
cells. The synthesis of oxaloacetate is a key step towards the
synthesis of succinate. In wild-type E. coli phosphoenol pyruvate
carboxylase represents the principle anaplerotic reaction to
replenish oxaloacetate. Under anaerobic conditions the portion of
phosphoenolpyruvate not flowing to oxaloacetate is converted to
pyruvate. In strains not expressing the heterologous pyruvate
carboxylase, pyruvate was observed to accumulate and succinate
yield decreased compared to the strain overexpressing pyruvate
carboxylase.
[0013] Lin et al (2005) have shown that the highest level of
succinate production in E. coli can be achieved by expressing both
phosphoenol pyruvate carboxylase from Sorghum vulgare and pyruvate
carboxylase from Lactococcus lactis when compared to E. coli
strains individually overexpressing either phosphoenol pyruvate
carboxylase or pyruvate carboxylase.
[0014] As indicated by these studies, all the efforts so far have
been focused on increasing the succinic acid production capability
by means of effectively utilizing the inorganic carbon already
present within the cell. This present invention provides a novel
method for enhancing the inorganic carbon uptake by bacterial cells
leading to an increase in the concentration of inorganic carbon
within the bacterial cell with the ultimate goal of increasing the
succinic acid production.
[0015] Generally, the inorganic carbon requirement for the
fermentative production of succinic acid is supplied either in the
form gaseous carbon dioxide or in the form of a carbonate salt such
as sodium carbonate, sodium bicarbonate, ammonium carbonate, and
ammonium bicarbonate. A number of US patents have disclosed the use
of inorganic carbon either to maintain the pH of the culture medium
or to maintain the growth rate of the microorganism. For example,
U.S. Pat. No. 5,958,744 uses NaHCO.sub.3 to neutralize the succinic
acid produced by the E. coli strain AFP 111. The sodium bicarbonate
addition to the fermentation medium besides maintaining the neutral
pH, also serves as a source of inorganic carbon required for the
carboxylation reactions within the cell. Andersson (2007) has
demonstrated that the use of Na.sub.2CO.sub.3 as a neutralizing
agent is desirable over the use of NH.sub.4OH, KOH, and NaOH as
neutralizing agents. It has been reported that NH.sub.4OH as a
neutralizing agent is toxic to E. coli and could cause a decrease
in the viability of the cells and the succinate productivity
(Andersson et al., 2009). Thus the prior art teaches away from the
use of NH.sub.4OH as the neutralizing agent in the succinic acid
production.
[0016] Andersson et al (2007) have disclosed the use of gaseous
carbon dioxide in the production of succinic acid using the
metabolically engineered E. coli strains AFP 111 and AFP184. These
succinic acid producing strains were grown in a medium maintained
at pH between 6.6 and 6.7 with the addition of NH.sub.4OH as 15%
NH.sub.3 solution. The anaerobic production phase was initiated by
withdrawing the air supply and sparging the culture medium with
CO.sub.2 at a flow rate of 3 L min.sup.-1.
[0017] U.S. Pat. No. 5,168,055 discloses that the growth conditions
for succinic acid producing Anaerospirillum succiniproducens
requires at least about 0.1 atmospheric CO.sub.2. The medium can be
sparged with CO.sub.2 gas. The fermentation can be run in a
pressurized reactor which contains CO.sub.2 at super atmospheric
pressure. The CO.sub.2 can be mixed with other gases as long as the
gases employed do not interfere with the growth. Carbon dioxide can
also be supplied to the fermentation medium by the addition of
carbonate or bicarbonate salts which generates CO.sub.2 gas under
the conditions of the fermentation. For sufficient succinic acid
production, the medium should contain dissolved CO.sub.2 in
equilibrium.
[0018] Promising succinic acid producing bacteria Mannheimia
succinciproducens and Actinobacillus succinogens have been isolated
from bovine rumen. The major gas produced in the rumen of the
cattle is CO.sub.2 (65.5 mol %). These strains of rumen bacteria
are capnophilic (CO.sub.2 loving) and produce succinic acid as the
major product from various carbon sources under 100% CO.sub.2
conditions at pH of 6.0 to 7.5. Genome-scale metabolic flux
analysis indicated that CO.sub.2 is important for the carboxylation
of phosphoenolpyruvate to oxaloacetate, which is converted to
succinic acid by the reductive tricarboxylic acid cycle (Lee et
al., 2002; Hong et al., 2004; Song and Lee., 2006).
[0019] Song et al (2007) have shown that in the capnophilic rumen
bacterium M. succiniproducens the production of succinic acid by a
carboxylation reaction during fermentation is dependent on
intracellular CO.sub.2. They investigated the metabolic responses
of M. succiniproducens to the different dissolved CO.sub.2
concentrations (0-260 mM). Cell growth was severely suppressed when
the dissolved CO.sub.2 concentration was below 8.74 mM. The cell
growth and succinic acid production increased proportionally as the
dissolved CO.sub.2 concentration increased from 8.74 to 141 mM. The
yields of biomass and succinic acid on glucose obtained at the
dissolved CO.sub.2 concentration of 141 mM were 1.49 and 1.52 times
higher respectively, than those obtained at the dissolved CO.sub.2
concentration of 8.74 mM. It was also found that the addition of
CO.sub.2 source provided in the form of NaHCO.sub.3, MgCO.sub.3, or
CaCO.sub.3 had positive effects on cell growth and succinic acid
production. However, growth inhibition was observed when excessive
bicarbonate salts were added. By the comparison of the activities
of key enzymes, it was found that phosphoenol pyruvate
carboxylation by phosphoenol pyruvate carboxykinase is most
important for succinic acid production as well as the growth of M.
succiniproducens by providing additional ATP.
[0020] U.S. Pat. No. 7,223,576 discloses the use of both sodium
bicarbonate and gaseous carbon dioxide in the production of
succinic acid by a mutant E. coli strain with the heterologous
pyruvate carboxylase gene from Lactococcus lactis. The pH of the
growth medium was maintained with 1.0 M Na.sub.2CO.sub.3 and
CO.sub.2 gas was sparged through the culture during the
fermentation period at a constant flow rate. The heterologus
expression of pyruvate carboxylase in a succinate producing strain
of E. coli increases the carbon flux from pyruvate to oxaloacetic
acid. Pyruvate carboxylase diverts pyruvate toward oxaloacetic acid
to favor succinate generation.
[0021] U.S. Pat. No. 7,244,610 discloses the aerobic succinate
production using a bacterial catalyst. The growth medium contained
2 g/L NaHCO.sub.3 and approximately 60 mM glucose. NaHCO.sub.3 was
added to the culture medium because it yielded better cell growth
and succinate production due to its pH-buffering capacity and its
ability to supply CO.sub.2.
[0022] U.S. Pat. No. 7,262,046 discloses a growth medium containing
2 g/L NaHCO.sub.3 in the aerobic succinate production using a
bacterial biocatalysts. The washed culture was then used to
inoculate a bioreactor containing LB with 2 g/L NaHCO.sub.3.
[0023] US Patent Application Publication No. 2006/0073577 A1
discloses the use of LB broth medium supplemented with 20 g/L of
glucose, and 1 g/L of NaHCO.sub.3 in the production of succinate.
NaHCO.sub.3 was added to the culture medium because of its
pH-buffering capacity and its ability to supply CO.sub.2.
[0024] US Patent Application No. 2009/0186392 A1 discloses a method
of glycerol fermentation where pH and CO.sub.2 concentrations are
controlled to allow the fermentative metabolism of glycerol to
desired chemical precursors. CO.sub.2 concentrations were
inevitably linked to pH and went down as pH increased because
CO.sub.2 was converted to bicarbonate. By increasing CO.sub.2 to
20-30% the negative effects of increased pH above 7.0 could be
reduced. Improved glycerol fermentation was seen with pH 6.3 and
10% CO.sub.2, and with pH 7.5 and 20% CO.sub.2. Greater
concentrations of CO.sub.2 were also beneficial.
[0025] U.S. Pat. No. 7,256,016 discloses a recycling system for
manipulation of intracellular NADH availability. The anaerobic tube
experiments were performed using 40 ml or 45 ml glass vials with
open top caps and PTFE/silicone rubber septa. Each vial was filled
with 35 ml or 40 ml of LB medium supplemented with 20 g/L glucose,
100 mg/L kanamycin, 0 or 50 mM formate and 1 g/L NaHCO.sub.3 to
reduce the initial lag time that occurs under anaerobic
conditions.
[0026] In a dual phase growth pattern for production of succinate,
the bacterial culture is initially grown in an aerobic condition
and transferred to an anaerobic production phase. The succinate
production occurs during the anaerobic growth phase. No growth
occurs during the anaerobic process. Glucose consumption and
product formation rates were essentially constant under anaerobic
conditions and the process exhibits a metabolic
pseudo-steady-state. The anaerobic biocatlaytic process for the
production of succinic acid has been shown to consume carbon
dioxide under non-growing anaerobic conditions. Since CO.sub.2 is
incorporated into the carbon backbone as a result of the
carboxylation of phosphoenol pyruvate by phosphoenol pyruvate
carboxylase, it is hypothesized that different CO.sub.2
concentrations in the gas phase would impact the metabolic fluxes
and ultimately change the yield and rate of succinate generated.
The effect of CO.sub.2 on succinate production in dual-phase
Escherichia coli fermentation is well documented (Lu et al.,
2009).
[0027] International patent application WO 2009/083756 A1 published
under the Patent Cooperation Treaty provides a large scale
microbial culture method for producing succinic acid using a
recombinant bacteria containing an over expressed pyruvate
carboxylase gene. The culture is initially grown aerobically in a
medium devoid of any inorganic carbon. After the growth in the
aerobic environment, the bacterial culture is acclimatized to
oxygen lean condition wherein the oxygen concentration is brought
down to less than 5% oxygen in the reactor by means of purging the
with CO.sub.2 or CO.sub.2 mixed with an inert gas. The carbon
dioxide thus supplied provides the source of inorganic carbon
required by the pyruvate carboxylase enzyme.
[0028] In the experiments with E. coli stain AFP111, it has been
shown that when the concentration of CO.sub.2 in the gas phase is
increased from 0% to 50%, the succinate specific productivity
increased from 1.9 mg/gh to 225 mg/gh and the succinate yield
increased from 0.04 g/g to 0.75 g/g. Above 50% CO.sub.2
concentration in the medium, succinate production did not increase
further. A four-process explicit model to describe the CO.sub.2
transfer and utilization has predicted that at CO.sub.2
concentration below about 30-40%, the system becomes limited by gas
phase CO.sub.2, while at higher CO.sub.2 concentrations the system
is limited by phosphoenol pyruvate carboxylase enzyme kinetics. At
limiting CO.sub.2 concentrations, the succinic acid production can
be rate limited at different stages. The diffusion of CO.sub.2 from
the gas phase into the liquid phase may be limiting. As a result of
poor equilibrium, the concentration of the CO.sub.2 in the liquid
phase may be several folds lower than the concentration of CO.sub.2
in the gas phase. Another step in the availability of CO.sub.2 lies
at the transfer of the dissolved CO.sub.2 from the exterior liquid
phase to the interior of the biocatalysts. The diffusion of
dissolved CO.sub.2 through the cell membrane may be too slow. Even
the permeation of HCO.sub.3 through the cell membrane may be
insignificant. Once inside the cell, the CO.sub.2 is converted into
bicarbonate [HCO.sub.3.sup.-] form so that it can be used as a
substrate for the functioning of the phosphoenol pyruvate
carboxyalse. The conversion of CO.sub.2 to bicarbonate is mediated
by carbonic anhydrase (Lu et al., 2009).
[0029] U.S. Pat. No. 6,455,284 discloses a dual-phase E. coli
fermentation for the production of succinic acid. The E. coli
strain used in this study contained a polynucleotide sequence
encoding a pyruvate carboxylase operatively linked to a promoter,
wherein said polynucleotide sequence is expressed and produces an
enzymatically active pyruvate carboxylase which is able to
incorporate the inorganic carbon in the growth medium into the
succinic acid produced. E. coli cells were grown aerobically in
Luria-Bertani (LB) medium. Anaerobic fermentation were carried out
in 100 ml serum bottles with 50 ml LB medium supplemented with 20
g/L glucose and 40 g/L MgCO.sub.3. The fermentations were
terminated at 24 hours at which point the pH value of all
fermentations were approximately pH 6.7.
[0030] US Patent Application Publication No. 2007/0111294 provides
growth coupled succinate production in E. coli strains. All
experiments were performed using M9 minimal medium at pH 7.0 (6.78
g/L Na.sub.2HPO.sub.4, 3.0 g/L KH.sub.2PO.sub.4, 0.5 g/L NaCl, 1.0
g/L NH.sub.4Cl, 1 mM MgSO.sub.4, 0.1 mM CaCl.sub.2) supplemented
with 2 g/L glucose and 20 mM NaHCO.sub.3. The inorganic carbon
required for the succinic acid production was provided by
NaHCO.sub.3 in the medium.
[0031] U.S. Pat. No. 7,563,606 provides a method for producing
succinic acid using the bacterial strain Brevibacterium flavum
MJ-233. Brevibacterium flavum may be currently classified into
Corynebacterium glutamicum. These bacterial cells showed an
enhanced pyruvate carboxylase activity due to the presence of a
plasmid coding for the pyruvte carboxylase activity. The
neutralization was carried out by using magnesium carbonate and
magnesium hydroxide. Supplementing the magnesium carbonate either
with ammonium hydrogen carbonate or sodium hydrogen carbonate
enhanced the succinic acid production rate and yield. CO.sub.2 gas
was also provided to the fermentation vessel. Apparently, the
CO.sub.2 gas and various carbonate and bicarbonate salts acted as
the source of the inorganic carbon required for the action of
pyruvate carboxylase enzyme contributing the flow of carbon towards
succinic acid.
[0032] US Patent Application Publication Nos. 2006/0205048 and
2008/0293113 provide a method for producing succinic acid in a
medium containing carbonate ion, bicarbonate ion or carbon dioxide
gas and a bacterial strain containing enhanced levels of pyruvate
carboxlase enzyme. The suitable bacterial strains are derived from
a group consisting of Coryneform bacterium, Bacillus bacterium, and
Rhizopium bacterium.
[0033] As described above, each of the microbial catalyst currently
in use for the production of succinic acid is known to require a
source of inorganic carbon for efficient production of succinic
acid. In view of the importance of the inorganic carbon in the
production of succinic acid, the present invention provides a novel
method for preparing solid inorganic carbonate and bicarbonate
salts by means of sequestering the carbon released from various
industrial applications. The carbon released from fossil fuel
burning and the operation of fermentation facilities can be trapped
in alkali solutions and the resulting carbonate and bicarbonate
salts can be used as a source of inorganic carbon in the
fermentative production of succinic acid. In addition, the present
invention provides a method for using the product resulting from
the sequestration of carbon dioxide.
BRIEF SUMMARY OF THE INVENTION
[0034] This invention is applicable to all industrial
microbiological process wherein the productivity and the yield of
end products are dependent on the uptake and utilization of
inorganic carbon from the medium. In particular, the present
invention is suitable for the production of organic acid thorough
anaerobic fermentation process. More specifically, the present
invention is useful in reducing the cost of production of succinic
acid through anaerobic fermentation process and in helping the
global carbon sequestration efforts.
[0035] In one embodiment, the present invention provides a method
for sequestering the carbon dioxide released during the
fermentation process. The utilization of carbon dioxide gas as a
source of inorganic carbon in the fermentation solution is very
inefficient. The solubility of carbon dioxide in aqueous solution
is several folds lower when compared to the concentration in the
gas phase. Moreover, the continuous pumping of carbon dioxide into
the fermentation vessel results in the release of carbon dioxide
into the atmosphere. The present invention overcomes this
limitation by means of supplying the required inorganic carbon in
the form of carbonate or bicarbonate salts which is obtained by
trapping the carbon dioxide gas released from the fermentation
vessel in alkali solution.
[0036] In one aspect, the present invention provides a cost
effective carbonate or bicarbonate salts suitable for the
biological production of succinic acid. The present invention shows
that the expensive K.sub.2CO.sub.3 and KOH used in the fermentation
process can be replaced with relatively inexpensive NH.sub.4OH. In
another aspect, the present invention provides a means of cost
saving by means of using NH.sub.4HCO.sub.3 as a source of inorganic
carbon in place of K.sub.2CO.sub.3 and KHCO.sub.3.
NH.sub.4HCO.sub.3 besides serving as a source of inorganic carbon
can also act as a source of nitrogen.
[0037] In yet another embodiment of the present invention, the
microbial culture is provided with microaeration during the
production phase of its growth. In one aspect of the present
invention, microaeration is provided in order to assure that there
is a complete consumption of the organic carbon supply in the
medium. In another aspect of the present invention, an appropriate
amount of microaeration is provided to increase the titer and the
productivity of succinic acid.
[0038] These fermentation process improvements can be utilized both
in the batch mode of fermentation and fed-batch mode of
fermentation. Moreover, these fermentation process improvements can
be practiced with a variety of microbial biocatalyst utilizing
starch and lignocellulosic hydrolysates derived from renewable
resources.
[0039] Additional advantage of this invention will become readily
apparent from the ensuing description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0040] FIG. 1. Process flow diagram for generating bicarbonate
salts useful as a source of inorganic carbon in the fermentative
production of succinic acid. As explained in Example 1, any one of
the commonly available alkali solutions such as ammonium hydroxide,
sodium hydroxide or potassium hydroxide is added to a vessel and
carbon dioxide gas is micro-sparged. The alkali solution inside the
container is stirred at 500 rpm for an hour. At the end of one
hour, the resulting white slimy liquid is appropriately diluted and
used in the microbial fermentation of organic carbon compounds
leading to the production of succinic acid.
[0041] FIG. 2. Effect of increasing the bicarbonate concentration
in the fermentation medium on succinic acid titer. The succinic
acid titer in grams per liter (g/L) of fermentation broth is shown
on the Y-axis. Shown on the x-axis is the % Molar bicarbonate
concentration in the fermentation medium. The % Molar bicarbonate
is the percentage of molar concentration of ammonium bicarbonate
with reference to the molar concentration of total ammonium
compounds present in the fermentation medium and include ammonium
hydroxide used for neutralization and ammonium bicarbonate acting
as a source of inorganic carbon.
[0042] FIG. 3. Effect of increasing bicarbonate concentration in
the fermentation medium on the molar ratio between acetic acid and
succinic acid production. The values on the Y-axis are the molar
ratio between acetic acid and succinic acid during fermentation in
the presence of varying concentration of bicarbonate. Shown on the
x-axis is the % Molar bicarbonate concentration in the fermentation
medium. The % Molar bicarbonate is the percentage of molar
concentration of ammonium bicarbonate with reference to the molar
concentration of total ammonium compounds present in the
fermentation medium and include ammonium hydroxide used for
neutralization and ammonium bicarbonate acting as a source of
inorganic carbon.
[0043] FIG. 4. Normalized succinic acid titer in grams/liter (g/L)
in the fermentation medium containing 100 mM NH.sub.4HCO.sub.3 and
10 mM KCl (solid line) or 100 mM KHCO.sub.3 (broken line). The
fermentation was run for a period of 36 hours.
[0044] FIG. 5. Kinetics of glucose utilization in the fermentation
medium containing 100 mM NH.sub.4HCO.sub.3 and 10 mM KCl (solid
line) or 100 mM KHCO.sub.3 (broken line). The glucose concentration
in the growth medium is expressed as grams/liter (g/L). The
fermentation was run for a period of 36 hours.
[0045] FIG. 6. Normalized cumulative succinic acid productivity in
the fermentation medium containing 100 mM NH.sub.4HCO.sub.3 and 10
mM KCl (solid line) or 100 mM KHCO.sub.3 (broken line). The
normalized cumulative succinic acid productivity is expressed in
terms of grams of succinic acid produced per liter per hour
(g/L/hr). The fermentation was run for a period of 36 hours.
[0046] FIG. 7. Titer (g/L) for succinic acid, acetic acid, pyruvic
acid, malic acid, and lactic acid in the fermentation medium
containing 100 mM KHCO.sub.3 as a source of potassium and inorganic
carabon. The fermentation was run for a period of 36 hours.
[0047] FIG. 8. Titer (g/L) for succinic acid, acetic acid, pyruvic
acid, malic acid, and lactic acid in the fermentation medium
containing 100 mM NH.sub.4CO.sub.3 as a source of inorganic carbon
and 10 mM KCl as a source of potassium. The fermentation was run
for a period of 36 hours.
[0048] FIG. 9. Kinetics of production of succinic acid under
fed-batch mode (solid line) and batch mode (broken line) of
fermentation. 7N NH.sub.4OH and 3M NH4HCO.sub.3 were used as the
source of neutralizing agent and source of inorganic carbon
respectively. KJ122 strain of E. coli was used as the
biocatalyst.
[0049] FIG. 10. The molar ratio between acetic acid and succinic
acid production during fed-batch mode (solid line) and batch mode
(broken line) of succinic acid fermentation. 7N NH.sub.4OH and 3M
NH.sub.4HCO.sub.3 were used as the source of neutralizing agent and
source of inorganic carbon respectively. KJ122 strain of E. coli
was used as the biocatalyst. Under fed-batch mode of fermentation,
the ratio of acetic acid produced to succinic acid produced was
found to be lower during most of production phase indicating the
production of acetic acid as a byproduct is much lower under
fed-batch mode of fermentation when compared to the acetic
production under batch mode of fermentation.
[0050] FIG. 11. Kinetics of glucose consumption in the control
succinic acid fermentation maintained under anaerobic condition
(solid line) and microaerated succinic acid fermentation (broken
line). The fermentations were carried out for 36 hours. At the end
of 36 hours of fermentation, glucose was completely consumed in the
microaerated samples while nearly 20 g/L of glucose remained in the
fermentation samples maintained under anaerobic condition. Ammonium
hydroxide and ammonium bicarbonate were used as neutralizing and
source of inorganic carbon respectively. Microaeration was provided
by supplying air at the rate of 0.1 vvm.
[0051] FIG. 12. Normalized cumulative succinic acid productivity
(g/L/hr) in the fermentation conducted under strict anaerobic
conditions (broken line) and in the fermentation conducted under
microaerobic condition (solid line). The fermentation was conducted
for 36 hours. Ammonium hydroxide and ammonium bicarbonate were used
as neutralizing and source of inorganic carbon respectively.
Microaeration was provided by supplying air at the rate of 0.1
vvm.
[0052] FIG. 13. Normalized succinic acid titer (g/L) in the
succinic acid fermentations aerated with different amounts of air
mixed with carbon dioxide gas. The fermentor was supplied with
carbon dioxide gas mixed with air at 3% (dotted line), 2% (broken
line) or at 0.5% (solid line). Ammonium hydroxide was used as
neutralizing agent.
[0053] FIG. 14. Normalized cumulative succinic acid productivity
(g/L/hr) in the succinic acid fermentations aerated with different
amounts of air mixed with carbon dioxide gas. The carbon dioxide
supply to fermentor was mixed with air at 3% (dotted line), 2%
(broken line) or at 0.5% (solid line). Ammonium hydroxide was used
as neutralizing agent.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0054] The present invention provides a process for the production
of organic acids in commercially significant quantities from the
fermentation of carbon compounds by recombinant microorganisms.
More specifically, this present invention provides the fermentation
parameters and the biocatalysts suitable for the production of
succinic acid at a higher yield. The biocatalysts and the
fermentation parameters of the present invention also result in an
increased productivity of the succinic acid. The term "yield" as
defined in this invention refers to ratio of grams of organic acid
produced per gram of organic carbon consumed. The term
"productivity" as defined in this invention refers to the actual
yield of succinic acid produced and is expressed in terms of number
of grams of organic acid produced per liter per hour. The term
"normalized yield" as defined in this invention refers to the ratio
of moles of organic acid produced per mole of organic carbon
consumed wherein the ratio is determined after factoring the
dilution that results from the addition of neutralizing agent and
other reagents to the fermentor. The biocatalysts of the present
invention possess the ability to enhance the inorganic carbon
uptake and utilization besides having ability to use multiple
sugars in the fermentation process for the production of
commercially significant quantities of organic acid.
[0055] The enhanced ability for inorganic carbon uptake can be
achieved by means of genetic modification. Genetic modification
leading to an enhanced ability for inorganic carbon uptake involves
introducing genes coding for bicarbonate transporters form
eukaryotic and prokaryotic photosynthetic microorganism into the
biocatalysts developed for the production of succinic acid (Price
at al., 2008; Spalding, 2008). For examples, the genes for
bicarbonate transporters can be introduced into the KJ122 strain of
E. coli developed for the commercial production of succinic acid.
The genes coding for bicarbonate transporters can be introduced
into the KJ122 strain in the form of a self-replicating plasmid
with genes coding for the bicarbonate transport proteins under the
control of promoters functional in the KJ122 strain. Alternatively,
the genes coding for bicarbonate transporters can be integrated
into the chromosomal DNA of KJ122 under the control of promoter
functional in KJ122.
[0056] The enhanced ability for utilizing inorganic acid can be
achieved by means of genetic modifications leading to an enhanced
activity of one or more carboxylating enzymes within the
biocatalysts selected for the commercial production of succinic
acid. In a preferred embodiment, the activity of one of the
carboxylating enzyme present within the biocatalyst selected for
succinic acid production is enhanced by genetic manipulations. For
example, the activity of the phosphoenol pyruvate carboxykinase
enzyme present within the E. coli based bacterial biocatalyst can
be enhanced by mean of introducing mutations in the promoter region
of the gene coding for this enzyme. Alternatively, the genes coding
for carboxylating enzymes such as pyruvate carboxylase or
phosphoenolpyruvate carboxyalse can be derived from exogenous
sources and introduced into the bacterial biocatalysts selected for
the production of succinic acid.
[0057] The requirement for inorganic carbon in the fermentative
production of organic acids such as succinic acid is now well
established and the required inorganic carbon can be supplied
either in the form of pure gaseous carbon dioxide or carbon dioxide
gas mixed with other gases. The carbon dioxide gas either alone or
mixed with other gases can be sparged through the fermentation
fluid. Alternatively, the inorganic carbon can be supplied in the
form of carbonate or bicarbonate salts of various alkali and
alkaline earth metals such as K.sub.2CO.sub.3, KHCO.sub.3,
Na.sub.2CO.sub.3, NaHCO.sub.3, (NH.sub.3).sub.2CO.sub.3 and
NH.sub.4HCO.sub.3. It is well known in the art that depending on
the pH of the medium, there is definite ratio between the
CO.sub.3.sup.-2, HCO.sub.3.sup.-1, and H.sub.2CO.sub.3 and the
corresponding cations. In a preferred embodiment, NH.sub.4HCO.sub.3
is used as the source of inorganic carbon. As used in the present
invention, the terms ammonium bicarbonate and ammonium hydrogen
carbonate are synonyms.
[0058] The supply of inorganic carbon in the form of carbonate or
bicarbonate salts of alkali and alkaline earth metals is preferred
over the supply of gaseous carbon dioxide as the solid form of
inorganic carbon increases the inorganic carbon concentration in
the fermentation medium beyond what could be achieved by the
continuous supply of carbon dioxide gas to the fermentation medium
in a cost effective way. Moreover, the use of bicarbonate salt in
place of gas phase CO.sub.2 also eliminates the issue related to
the poor diffusion of CO.sub.2 from the gas phase into the aqueous
phase.
[0059] The solid form of inorganic carbon such as carbonate and
bicarbonate salts of alkali metal and alkaline earth metals
required for the fermentative production of organic acid may be
obtained from commercial sources. In a preferred embodiment, the
solid inorganic carbon source is prepared by means of sparging
carbon dioxide containing gas through an alkali solution. Either a
pure carbon dioxide gas or a flue gas emanating from fossil fuel
based power generators or waste gases from large scale industrial
fermentation tanks can be sparged through the alkali solutions such
as ammonium hydroxide, potassium hydroxide, magnesium hydroxide and
sodium hydroxide under pressure and constant stirring till
precipitation begins (FIG. 1). In a preferred embodiment, the
carbon dioxide emanating from the fermentation vessel is pumped
through the ammonium hydroxide solution. Trapping the carbon
dioxide gas coming from the fermentation vessel in an alkali
solution and utilizing the resulting carbonate salts as a source of
inorganic carbon in the fermentation constitutes a part of an
integrated fermentation facility. In the most preferred embodiment,
the carbon dioxide gas emanating from a fermentation facility is
sparged into a tank containing 19%-28% NH.sub.4OH solution leading
to the production of saturated solution of ammonium bicarbonate.
The saturation level of ammonium bicarbonate solution is reached
when the concentration of ammonium bicarbonate in the solution
reached approximately 3M. By means of starting with ammonium
hydroxide solution of different molar concentrations, it is
possible to obtain solutions containing specific ratio for ammonium
hydroxide and ammonium bicarbonate.
[0060] The carbon dioxide used for the preparation of inorganic
carbon solids can be derived from the same fermentation vessel
where the carbonate salt resulting from carbon dioxide capture ends
up. Alternatively, the carbon dioxide gas can be derived from a
different fermentation vessel or from a different fermentation
plant. For example, the carbon dioxide gas released from an ethanol
plant can be captured in an alkali solution and the resulting solid
carbonate can be used as a source of inorganic carbon in a succinic
acid plant. Even aerobic fermentations such as those producing
antibiotics and vitamins and releasing both oxygen and carbon
dioxide could be utilized because carbon dioxide would be trapped
in the alkali solution while oxygen would leave the alkali trap.
The list of the solid inorganic carbons suitable for the present
invention includes sodium carbonate, sodium bicarbonate, potassium
carbonate, potassium bicarbonate, magnesium carbonate, magnesium
bicarbonate, ammonium carbonate, and ammonium bicarbonate. Among
these solid inorganic carbon compounds, ammonium carbonate and
ammonium bicarbonate are preferred inorganic carbon sources.
[0061] In the commercial scale manufacturing of organic acid using
biological feedstock and inorganic carbon sources, it is necessary
to control the inhibitory effect of organic acid being produced on
the viability of the bacterial cells. Therefore it is necessary to
add a neutralizing agent to the culture medium for the purpose of
neutralizing the organic acid being produced. The pH of the culture
vessel can be continuously monitored using a pH probe, and
appropriate base can be added to maintain the pH of the growth
medium around neutral pH. The list of bases suitable for
maintaining the pH of the microbial culture includes, but not
limited to NaOH, KOH, NH.sub.4OH, Mg(OH).sub.2, Na.sub.2CO.sub.3,
NaHCO.sub.3, and (NH.sub.4)HCO.sub.3, (NH.sub.4).sub.2CO.sub.3. The
bases suitable for this purpose can be used alone or in
combination. In a preferred embodiment, the alkali solution used to
trap the gaseous carbon in the production of solid inorganic carbon
can also act as a neutralizing agent.
[0062] One requirement in selecting the neutralizing base in the
commercial scale manufacturing of organic acid is to select a base
which is low-cost and compatible with the biocatalysts being used
and the recovery process for organic acid. At laboratory scale, the
succinic acid neutralization has been achieved using a combination
of 1.2 M KOH and 2.4 M K.sub.2CO.sub.3. The potassium salts are too
expensive to use in the large scale commercial manufacturing. Since
NaOH is a low-price commodity chemical, sodium bases such as NaOH
and Na.sub.2CO.sub.3 are preferred neutralizing bases for the large
commercial scale manufacturing of organic acid through biological
fermentation. According to the present invention, ammonium
hydroxide is the most preferred base for maintaining the pH of the
fermentation vessel due to low cost and for the other reasons given
below.
[0063] Ammonium succinate is accumulated when the NH.sub.4OH and
NH.sub.4HCO.sub.3 are used as the source of neturalizing base and
the source of inorganic carbon respectively in the fermentation
medium for the production of succinic acid. The ammonium succinate
resulting from the use NH.sub.4HCO3 and NH.sub.4OH is treated with
sulfuric acid in the recovery of succinic acid with the resulting
formation of ammonium sulfate as a byproduct. Alternatively, the
ammonium succinate solution can be passed through an ion-exchange
resin and split into succinic acid and an ammonium salt. The large
volume ammonium sulfate byproduct resulting from the commercial
manufacture of succinic acid can be sold as a fertilizer and
thereby account for a significant cost recovery. With the
replacement of K.sub.2CO.sub.3 and KOH by NH.sub.4HCO.sub.3 and
NH.sub.4OH, the cost of the neutralizing agent for producing a
pound of succinic acid is reduced substantially as the price of
NH.sub.4OH in the commercial market is much lower than KOH and
K.sub.2CO3. It is also possible to synthesize NH.sub.4HCO.sub.3 at
the manufacturing facility using CO.sub.2 gas and NH.sub.4OH
solution at much cheaper cost and thereby adding further cost
savings.
[0064] This observation that NH.sub.4HCO.sub.3 and NH.sub.4OH can
be used as effectively as KOH/K.sub.2CO3 as a neutralizing agent is
in contrast to the prior art teaching against the use of NH.sub.4OH
as the neutralizing agent in succinic acid production. It has been
reported that NH.sub.4OH is toxic to E. coli and it could cause a
decrease in the viability of the cells and the succinate
productivity (Andersson et al., 2009).
[0065] As a neutralizing agent, NH.sub.4OH is used in the
concentration range of 1M to 15M. In the preferred embodiment,
NH.sub.4OH is used in the concentration range of 2M to 9M. In the
most preferred embodiment, NH.sub.4OH is used in the concentration
range of 6M to 8M.
[0066] Along with NH.sub.4OH as the neutralizing agent, a source of
inorganic carbon is also provided. Any of the commercially
available inorganic carbonate or bicarbonate salts can be used as a
source of inorganic carbon. The inorganic salts useful as a source
of inorganic carbon include sodium carbonate, sodium bicarbonate,
magnesium carbonate, magnesium bicarbonate, potassium carbonate,
potassium bicarbonate, ammonium carbonate, and ammonium
bicarbonate. The bicarbonate salts are preferred over the carbonate
salts. Among the bicarbonate salts, ammonium bicarbonate is
preferred for the reasons of cost saving and cost recovery.
[0067] The inorganic carbonate and bicarbonate salts can be used in
the range of 0.1 M to 6 M. In the preferred embodiment,
NH.sub.4HCO.sub.3 is used in the concentration range of 0.1M to 5
M. In the most preferred embodiment, NH.sub.4HCO.sub.3 is used in
the concentration range of 2 M to 4 M. The molar ratio between
NH.sub.4OH and NH.sub.4HCO.sub.3 is in the range of 8:1 to 1:1. The
preferred molar ratio between NH.sub.4OH and NH.sub.4HCO.sub.3 is
in the ration of 6:1 to 2:1. The most preferred molar ratio between
NH.sub.4OH and NH.sub.4HCO.sub.3 is in the ratio of 3:1 to 2:1. The
ratio of 8:3 between NH.sub.4OH and NH.sub.4HCO.sub.3 is the most
preferred in the production of succinic acid using bacterial
biocatalysts.
[0068] The ammonium hydroxide and ammonium bicarbonate solution can
be prepared separately and supplied to the fermentor independent of
each other. Ammonium hydroxide is supplied when it is required to
maintain the pH of the fermentation medium in the near neutral
range. Ammonium bicarbonate solution can be added at the beginning
or supplied when required. In a preferred embodiment, the ammonium
hydroxide and ammonium bicarbonate solution are preferred as a
single combined solution and added to the fermentor when it is
required to maintain the pH of the fermentation medium.
[0069] The mixture of NH.sub.4OH and NH.sub.4HCO.sub.3 can be
prepared by dissolving ammonium bicarbonate salt in the ammonium
hydroxide solution. In a preferred embodiment, the mixture of
NH.sub.4OH and NH.sub.4HCO.sub.3 can be prepared by means of
capturing carbon dioxide gas emanating from any industrial facility
and there by contributing to the global efforts towards reducing
carbon emission through carbon sequestration.
[0070] It is also possible to supplement the addition of solid
inorganic carbon to the medium with a supply of carbon dioxide gas.
The carbon dioxide can be sparged through the fermentor at a rate
of 0.01 volume per volume per minute (vvm) to 1.0 vvm. In a
preferred embodiment, the carbon dioxide gas is applied at the rate
of 0.05 vvm to 0.5 vvm. In the most preferred embodiment, the
carbon dioxide gas is applied at the rate of 0.1 vvm.
[0071] The list of the bacterial species suitable for development
as a biocatalyst for the fermentative production of organic acids
according to this invention includes Escherichia coli,
Gluconobacter oxydans, Gluconobacter asaii, Achromobacter
delmarvae, Achromobacter viscosus, Achromobacter lacticum,
Agrobacterium tumefaciens, Agrobacterium radiobacter, Alcaligenes
faecalis, Arthrobacter citreus, Arthrobacter tumescens,
Arthrobacter paraffineus, Arthrobacter hydrocarboglutamicus,
Arthrobacter oxydans, Aureobacterium saperdae, Azotobacter indicus,
Brevibacterium ammoniagenes, divaricatum, Brevibacterium
lactofermentum, Brevibacterium flavum, Brevibacterium globosum,
Brevibacterium fuscum, Brevibacterium ketoglutamicum,
Brevibacterium helcolum, Brevibacterium pusillum, Brevibacterium
testaceum, Brevibacterium roseum, Brevibacterium immariophilium,
Brevibacterium linens, Brevibacterium protopharmiae,
Corynebacterium acetophilum, Corynebacterium glutamicum,
Corynebacterium callunae, Corynebacterium acetoacidophilum,
Corynebacterium acetoglutamicum, Enterobacter aerogenes, Erwinia
amylovora, Erwinia carotovora, Erwinia herbicola, Erwinia
chrysanthemi, Flavobacterium peregrinum, Flavobacterium fucatum,
Flavobacterium aurantinum, Flavobacterium rhenanum, Flavobacterium
sewanense, Flavobacterium breve, Flavobacterium meningosepticum,
Micrococcus sp. CCM825, Morganella morganii, Nocardia opaca,
Nocardia rugosa, Planococcus eucinatus, Proteus rettgeri,
Propionibacterium shermanii, Pseudomonas synxantha, Pseudomonas
azotoformans, Pseudomonas fluorescens, Pseudomonas ovalis,
Pseudomonas stutzeri, Pseudomonas acidovolans, Pseudomonas
mucidolens, Pseudomonas testosteroni, Pseudomonas aeruginosa,
Rhodococcus erythropolis, Rhodococcus rhodochrous, Rhodococcus sp.
ATCC 15592, Rhodococcus sp. ATCC 19070, Sporosarcina ureae,
Staphylococcus aureus, Vibrio metschnikovii, Vibrio tyrogenes,
Actinomadura madurae, Actinomyces violaceochromogenes,
Kitasatosporia parulosa, Streptomyces coelicolor, Streptomyces
flavelus, Streptomyces griseolus, Streptomyces lividans,
Streptomyces olivaceus, Streptomyces tanashiensis, Streptomyces
virginiae, Streptomyces antibioticus, Streptomyces cacaoi,
Streptomyces lavendulae, Streptomyces viridochromogenes, Aeromonas
salmonicida, Bacillus pumilus, Bacillus circulans, Bacillus
thiaminolyticus, Escherichia freundii, Microbacterium
ammoniaphilum, Serratia marcescens, Salmonella typhimurium,
Salmonella schottmulleri, Xanthomonas citri, Bacillus subtilis,
Bacillus licheniformis, Bacillus amylolliquefaciens and so forth.
The yeast species selected from the following genera are also
suitable for development of biocatalyst for the production of
organic acids including succinic acid: Saccharomyces,
Kluyveromyces, Candida, Zygosaccharomyces, Torulopsis, Torulospora,
Williopsis, Issatchenkia, Pichia, Schizosaccharomyces, Phaffia,
Cryptoccus, Yarrowia, and Saccharomycopsis. These strains of
microorganisms can be grown in the medium with a source of organic
carbon and inorganic carbon compounds as described here.
[0072] As defined in this invention, the term biocatalyst includes
microorganisms that have been developed for the purpose of
manufacturing organic acid including succinic acid using biological
feedstocks and inorganic carbon.
[0073] The microbial organisms of the present invention are grown
in a number of different culture medium well known in the field of
microbiology. For example, different strains of E. coli selected
for succinic acid production are grown in Luria-Bertani (LB) medium
containing 1% (w/v) tryptone, 0.5% (w/v) yeast extract, and 0.5%
(w/v) NaCl. For the commercial production of the organic acid using
fermentative processes involving a genetically modified
microorganism as biocatalyst, a minimal mineral salt medium
supplemented with a carbon source is preferred. The use of a
minimal mineral salt medium as opposed to a rich medium like LB
medium reduces the cost for the production of organic acids in a
commercial scale. The minimal mineral mediums suitable for the
present invention include NBS medium (Causey et al., 2007) and AM1
medium (Martinez et al., 2007). The NBS medium contains 1 mM
betaine, 25.72 mM KH.sub.2PO.sub.4, 28.71 mM K.sub.2HPO.sub.4,
26.50 mM (NH.sub.4)2HPO.sub.4, 1 mM MgSO.sub.4.7H.sub.2O, 0.1 mM
CaCl.sub.2.2H.sub.2O, 0.15 mM Thiamine HCl, 5.92 .mu.M
FeCl.sub.36H.sub.2O, 0.84 .mu.M CoCl.sub.2.6H.sub.2O, 0.59 .mu.M
CuCl.sub.2. H.sub.2O, 1.47 .mu.M ZnCl.sub.2, 0.83 .mu.M
Na.sub.2MoO.sub.42H.sub.2O, and 0.81 .mu.M H.sub.3BO.sub.3. The AM1
medium contains 1 mM betaine, 19.92 mM (NH.sub.4).sub.2HPO.sub.4,
7.56 mM NH.sub.4H.sub.2PO.sub.4, 1.5 mM MgSO.sub.4.7H.sub.2O, and
trace elements including 8.88 .mu.M FeCl.sub.36H.sub.2O, 1.26 .mu.M
CoCl.sub.2.6H.sub.2O, 0.88 .mu.M CuCl.sub.2.2H.sub.2O, 2.20 .mu.M
ZnCl.sub.2, 1.24 .mu.M Na.sub.2MoO.sub.42H.sub.2O, 1.21 .mu.M
H.sub.3BO.sub.3 and 2.50 .mu.M MnCl.sub.24H.sub.2O. Corn steep
liquor can be used in place of yeast extract and peptone. It is a
byproduct from the corn wet-milling industry. When compared to the
yeast extract and peptone, it is an inexpensive source of vitamins
and trace elements.
[0074] In certain bacterial fermentations, it is necessary to have
potassium ion in the growth medium. The potassium can be provided
either in the form of KCl, or KHCO.sub.3 or KH.sub.2PO.sub.4. These
potassium salts can be used in the range of 1 mM to 100 mM. It is
preferable to use KH.sub.2PO.sub.4 at 10 mM concentration for the
reasons of cost saving.
[0075] The mineral medium for microbial growth is supplemented with
both an organic and inorganic carbon source. Suitable fermentation
broths for use in the present process preferably include at least
about 20 g/L or at least about 30 g/L or at least about 40 g/L of
one or more carbohydrate and/or sugar containing sources. More
preferably, the fermentation broth includes at least about 70 g/L
and most preferably, at least 120 g/L of the carbohydrate and/or
sugar containing sources. The organic carbon sources useful in the
present invention include but not limited to pentose sugars like
xylose, and hexose sugars like glucose, fructose, galactose and
glycerol. The organic carbon source is also be satisfied by
providing a combination of different sugars such as a combination
of glucose and xylose. The carbon source can also be derived from a
hydrolysis of starch or lignocellulose. The hydrolysis of complex
carbohydrates such as starch and lignocelluloses is achieved either
by using thermo-chemical conversion processes or enzymatic methods
well known in the art. For example, the hexose sugars suitable for
the fermentation process of the present invention can be derived
from grain sorghum flour through enzyme digestion. The preferred
carbon source for the industrial production of organic acid using
microbial fermentation is lignocellulosic hydrolysate derived from
the hydrolysis of agricultural or forestry wastes. The
lignocellulosic hydrolysate is further fractionated to yield a
hexose-enriched and a pentose-enriched fraction and those fractions
serve as the source of carbon for the commercial production of the
organic acids using microbial fermentation processes. The
lignocellulosic hydrolysate is further detoxified to remove certain
chemicals such as furfural which are found to be toxic to a number
of microbial organisms above certain concentrations.
[0076] A nitrogen providing compound is also supplied in the
fermentation broth of the present invention as a nitrogen source
for the organic acid producing microorganism to begin growth and
start the fermentation process. Nitrogen producing compounds may
include ammonium phosphate, urea or any other suitable compound
containing nitrogen. The nitrogen producing compound may be present
by weight in an amount of between about 0.1% and 10%, and more
preferably between about 0.15% and 5%, and most preferably between
about 0.18% and 3%.
[0077] Fermentation reaction vessels of any suitable, known type
may be employed in performing the fermentation process of the
present invention. The size of the fermentors suitable for the
present invention is in the range of 3 L to 400,000 L. A variety of
reactor configurations including packed bed reactors, continuous
stirred tank reactors, rotating biological contact reactors,
sequencing batch reactors and fluidized bed reactors may be used in
the present process. The fermentation can be carried out by any
known methods in the field of industrial microbiology and
biotechnology. For example, the fermentation can be carried out in
a continuous process or a batch mode or a fed-batch mode. The
fed-batch mode of fermentor operation is preferred.
[0078] Further improvements in the yield and productivity of the
desired organic compounds in the microorganisms selected for
efficient carbon uptake and utilization capacities is achieved by
manipulating appropriate fermentation parameters. The
microorganisms suitable for the practice of the present invention
are grown aerobically (in the presence of oxygen) or anaerobically
(in the complete absence of oxygen). In one embodiment, the
microorganisms suitable for the present invention are grown in a
dual-phase growth regime, wherein the microorganism is initially
grown in aerobic growth condition to reach a certain level of cell
mass before transferring it to the anaerobic growth condition to
achieve the production of required organic acids in commercially
significant quantities. Cell mass was estimated by measuring the
optical density at 550 nm (OD.sub.550 nm) using a
spectrophotometer. During the production phase, the concentration
of dissolved oxygen is maintained at approximately zero. This can
be achieved either by means of sparging the fermentation vessel
with carbon dioxide or nitrogen gas. The dissolved oxygen
concentration is measured using a Clark-type oxygen electrode with
gas permeable membrane.
[0079] The inventors have surprisingly found that by means of
providing a minimal amount of oxygen during the production phase,
the yield and productivity of the organic compounds is further
improved. With the microaerobic condition during the production
phase, there is a better utilization of organic carbon present in
the medium as opposed to utilization of only 80% of the organic
carbon under strict anaerobic condition during the production
phase. The enhanced carbon utilization during microaerobic
production phase is further accompanied by a noticeable increase in
the yield and productivity of the organic compound.
[0080] The microaerobic condition can be achieved by means of
mixing the air in appropriate amount with a carrier gas.
Alternatively an appropriately low flow rate of air can be sparged.
The oxygen level in the fermentation fluid can be monitored using
an oxygen electrode or any other suitable device and the flow rate
of the gas mix is adjusted to assure that the level of oxygen in
the fermentation fluid is maintained at a constant level.
[0081] Microaeration rate suitable for the present invention is in
the range of 0.0001 vvm to 0.1 vvm, preferably from about 0.001 to
about 0.025 vvm, and even more preferably about 0.001 to about
0.0025 vvm with reference to the air used in the microaeration.
Aeration is preferably done under conditions such as sparging that
promotes the formation of fine gas bubbles. Agitation is preferably
maintained.
[0082] The concentration of various organic acids and sugars are
measured by HPLC. Succinic acid and other organic acids present in
the fermentation broth are analyzed on Agilent 1200 HPLC apparatus
with BioRad Aminex HPX-87H column. BioRad Microguard Cation H.sup.+
is used as a guard column. The standards for HPLC analysis are
prepared in 0.008N sulfuric acid. The HPLC column temperature is
maintained at 50.degree. C. Sulfuric acid at 0.008N concentration
is used as a mobile phase at the flow rate of 0.6 ml/min.
Quantification of various components is done by measuring their
absorption at 210 nm. The HPLC technology is also helpful in
determining the purity of the organic acid produced by the selected
clones.
Example 1
Preparation of Solid Inorganic Carbon Source
[0083] A stock solution containing both ammonium hydroxide and
ammonium bicarbonate was prepared by means of sequestering carbon
dioxide in the solution of ammonium hydroxide (FIG. 1). One liter
of 28-30% ammonium hydroxide solution was added to a 3 liter NBS
(New Brunswick Scientific) fermentor and carbon dioxide gas was
micro-sparged at the rate of 1 L/minute. The ammonium hydroxide
solution inside the fermentor was stirred at 500 rpm for an hour.
At the end of one hour, the temperature of the fermentor had
increased to 39.3.degree. C. from an initial temperature of
19.5.degree. C. Cooling of the fermentor was initiated by
circulating cold water through a coil within the fermentor. With
the cold water circulation the temperature of the fermentor reached
16.2.degree. C. in about 2 hours. When the solution turned into a
white slimy liquid, 325 ml of water was added to obtain
approximately 11 M combined solution of ammonium with approximately
3 M bicarbonate. 11 M concentration of combined ammonium
bicarbonate and ammonium hydroxide solution is an estimate based on
the initial volume of 1 L of 14.5 M NH.sub.4OH plus 325 ml of water
added to get the precipitated bicarbonate back into solution. It is
based on the assumption that the addition of CO.sub.2 did not
change the volume.
[0084] The combined ammonium hydroxide and ammonium bicarbonate
solution obtained as described above was used in the fermentation
of glucose in AM1 medium with the KJ122 strain of E. coli as a
biocatalyst for the production of succinic acid in a total volume
of 3,000 ml in a NBS fermentor maintained at 39.degree. C. The
fermentation medium also contained KH.sub.2PO.sub.4 (110 ml of 1M
KH.sub.2PO.sub.4). The concentration of ammonium hydroxide was
about 8N and the concentration of ammonium bicarbonate was about
3M. KJ122 inoculum had an initial OD.sub.550 nm of 7.8 and 150 ml
of this inoculum representing 5% (v/v) of the total fermentation
volume was used. The pH was maintained at 6.7 and fermentation
fluid was stirred with the impeller within the fermentor operated
at 750 RPM. Glucose solution at the concentration of 650 g/L was
fed as required. At the end of 36 hours of the production phase of
fermentation, the succinic acid titer was 93.2 g/L and yield was
0.86 gram of succinic acid per gram of glucose consumed. The titer
for acetic acid was 3.8 g/L.
Example 2
Succinic Acid Production with NH.sub.4OH and NH.sub.4HCO.sub.3
[0085] In order to identify the optimal ratio for NH.sub.4OH and
NH.sub.4HCO.sub.3 in the fermentative production of succinic acid,
a series of succinic acid fermentations were conducted with varying
ratios of NH.sub.4OH and NH.sub.4HCO.sub.3. As shown in the Table 1
below, twelve different NH.sub.4OH--NH.sub.4HCO.sub.3 compositions
were tested in the succinic acid fermentation using the KJ122
strain of E. coli as a biocatalyst in a total volume of 2,000 ml in
a NBS fermentor maintained at 39.degree. C. The fermentation medium
also contained KH.sub.2PO.sub.4 (55 ml of 1M KH.sub.2PO.sub.4),
MgSO.sub.4 (4 ml of 1.5 M MgSO.sub.4), betaine (4 ml of 1M
betaine). and trace elements. KJ122 inoculum had an initial
OD.sub.550 nm of 6.8 to 7.8 and 150 ml of this inoculum
representing 7.5% (v/v) of the total fermentation volume was used.
The pH was maintained at 6.5 and fermentation fluid was stirred
with the impeller within the fermentor operated at 750 RPM. Glucose
solution was fed as required. At the end of the fermentation the
titers for succinic acid and acetic acid as well as the succinic
acid yield were determined using HPLC technique. As the results
shown in FIG. 2 indicate, the succinic acid titer showed a linear
increase starting with the 10% molar bicarbonate concentration. The
succinic acid titer reached a plateau after about 20% molar
bicarbonate concentration. As defined in this invention, the %
molar bicarbonate is the percentage of molar concentration of
ammonium bicarbonate in the succinic acid fermentation medium with
reference to the molar concentration of total ammonium compounds
present in the fermentation medium. Thus if 8M NH.sub.4OH and 1M
NH.sub.4HCO.sub.3 are the only ammonium compounds present in the
fermentation medium, the % molar bicarbonate value is 1/9=0.111
(11.1%). Also measured in this experiment was the molar ratio
between the acetic acid and succinic acid in the fermentation broth
with reference to increase in the % molar bicarbonate concentration
in the fermentation medium. As the result shown in FIG. 3
indicates, once the succinic acid titer reaches a maximum value,
any further increase in the % molar bicarbonate value caused a
decrease in the titer for the acetic acid leading to a decrease in
the ratio of acetic acid to succinic acid in the fermentation
broth. The general observation from these fermentation runs was
that in contrast to the prior art teaching against the use of
NH.sub.4OH and NH.sub.4HCO3 as neutralizing agent and source of
inorganic carbon in the succinic acid production respectively,
commercially acceptable levels of succinic acid production was
achievable using NH.sub.4OH and NH.sub.4HCO.sub.3 in the
fermentations medium for succinic acid production.
Example 3
Potassium Requirement in Succinic Acid Production
[0086] In this study efforts were made to determine whether
potassium salts could be entirely eliminated from the fermentation
medium without any significant effect on the succinic acid
productivity. In the control experiment, the fermentation was
carried out with an initial volume of 4,000 ml in AM1 medium using
KJ122 as a biocatalyst. 3N NH.sub.4OH and 0.75 M K.sub.2CO.sub.3,
and 1.5 N KOH were used as the neutralizing base. Glucose was
provided at the initial concentration of 102.9 grams per liter. At
the end of 38 hours of fermentation, the glucose was completely
utilized. At the end of 38 hours of fermentation, the succinic acid
productivity was calculated to be 1.45 g/L/hr. In the second
experiment, fermentation was carried out with 6N NH.sub.4OH as the
only neutralizing base in an initial volume of 2,000 ml.
K.sub.2CO.sub.3 and KOH were completely eliminated from the
fermentation medium. Carbon dioxide gas was provided as the source
of inorganic carbon at the rate of 1 vvm (volume/volume/minute; 2
liters per minute). Glucose was provided at an initial
concentration of 98.4 grams/liter. At the end of 70 hours of
fermentation, the medium contained 2.8 grams of glucose/liter
suggesting that the glucose consumption was not complete even after
70 hours of fermentation when there was no potassium in the
fermentation medium. The succinic acid productivity for this
fermentation without any added potassium was found to be 0.84
g/L/hr.
[0087] Based on the result of the fermentation experiments
conducted without the addition of any potassium, another set of
fermentations were conducted to test the ability of 10 mM KCl to
satisfy the requirement for potassium in the succinic acid
fermentation. In this set of experiment the ability of 10 mM KCl to
replace 100 mM KHCO.sub.3 in the fermentation medium was tested. In
the control experiment, fermentation was carried out in an initial
volume of 3,000 ml in AM1 medium with 150 ml of 2M KHCO.sub.3 and
6N NH.sub.4OH was used as neutralizing agent. Glucose was added at
the initial concentration of 100 g/L. KJ122 strain of E. coli was
used as the biocatalyst and the fermentation was conducted for a
period of 36 hours. In another experiment conducted in parallel,
fermentation was carried out in an initial volume of 1,500 ml in
AM1 medium with 75 ml of 2M NH.sub.4HCO.sub.3 and 6N NH.sub.4OH was
used as the neutralizing agent. Glucose was added at the initial
concentration of 100 g/L. KJ122 strain of E. coli was used as the
biocatalyst and the fermentation was conducted for a period of 36
hours. Samples were drawn out from both the experiments and the
amount of glucose, succinic acid, acetic acid, pyruvic acid, malic
acid, and lactic acid in the fermentation broth were determined
using HPLC technique.
[0088] As the results shown in FIGS. 4 and 6 indicate succinic acid
titer and succinic acid productivity in the samples derived from
the fermentation runs containing 10 mM KCl and fermentation runs
containing 100 mM KHCO.sub.3 were very much comparable to each
other. Moreover, as the results shown in FIG. 5 indicates, the rate
of glucose consumption were also comparable between the
fermentation run with 10 mM KCl and the fermentation run with 100
mM KHCO.sub.3.
[0089] The results shown in FIGS. 7 and 8 indicate that the
composition of various organic acids produced in the fermentation
runs with 100 mM KHCO.sub.3 is very much comparable to the
fermentation run with 10 mM KCl. Thus the substitution of
KHCO.sub.3 with NH.sub.4HCO.sub.3 did not alter the titer for
succinic acid with reference to the titer of other organic acids
produced as byproducts.
Example 4
Comparison of Batch and Fed-Batch Fermentation
[0090] There are several advantages associated with operating the
fermentor in the fed-batch mode when compared to the batch mode
operation. In the fed-batch mode, the biocatalyst is subjected to
less osmotic stress as the sugar substrate is added gradually.
Moreover, it is possible to avoid any potential waste in the
organic carbon feedstock by means of feeding organic compounds only
when it is required under fed-batch mode. In the case of batch mode
of fermentation, the entire amount of organic carbon is added at
the beginning of the fermentation and when the fermentation does
not consume the organic carbon entirely, the left over organic
carbon is in the waste stream at the end of the fermentation run.
In order to determine whether the succinic acid production is
comparable both in the fed-batch and batch mode of fermentor
operations, parallel experiments were conducted both in the batch
mode and in the fed-batch mode. In the fed-batch, the fermentation
was conducted with an initial volume of 2,000 ml with 48 ml of 1M
KH.sub.2PO.sub.4, 3.5 ml of 1.5 MgSO.sub.4, 3. 5 ml of 1 M Betaine
and trace elements and neutralized with 7N NH.sub.4OH and 3 M
NH.sub.4HCO.sub.3. KJ122 biocatalyst was inoculated and the pH was
maintained at 6.5. The fermentation fluid was stirred by operating
the impeller within the fermentor at 750 rpm. The initial glucose
concentration was 25 g/l and additional glucose was fed as
required. In the fermentation conducted in the batch mode, the same
medium was batched in 4,000 ml. 150 ml KJ122 biocatalyst was
inoculated and the pH was maintained at 6.5. The fermentation fluid
was stirred by operating the impeller within the fermentor at 750
rpm. Glucose was added at the initial concentration of 100 g/l.
[0091] The productivity and the relative ratio between acetic acid
and succinic acid were measured under both fermentation conditions.
As the result shown in FIG. 9 indicates, the succinic acid titer
was comparable between batch and fed-batch modes of fermentation.
The succinic acid titer in the fed-batch fermentation was 85.2
gram/l while the titer for acetic acid was 3.2 g/l. On the other
hand, in the batch mode, the succinic acid titer was 67.1 g/l and
acetic acid titer was 5.3 g/L. The yield of succinic acid was 80.2
grams of succinic acid per gram of glucose consumed in the
fed-batch as compared to the succinic acid yield of 71.8 grams of
succinic acid per gram of glucose consumed in the batch mode. In
addition, it was surprisingly noticed that the acetic acid to
succinic acid ratio was lower in the fed-batch mode of fermentation
when compared to the acetic acid to succinic acid ratio in the
batch mode of fermentation (FIG. 10).
Example 5
Microaeration of Fermentation Vessel
[0092] Since glucose consumption in the absence of potassium does
not go to completion, efforts were made to determine whether
providing microaeration would enhance the fermentation. In the
control experiment, fermentation was carried out in a total volume
of 9,000 ml with 6N NH.sub.4OH as the neutralizing base. 100 mM
NH.sub.4HCO.sub.3 was provided as the source of inorganic carbon.
Additional inorganic carbon source was provides by supplying carbon
dioxide at the rate of 0.1 vvm. KJ122 biocatalyst was inoculated at
the initial OD.sub.550 nm of 6.2 and the pH was maintained at 6.75.
Fermentation fluid was stirred by operating the impeller within the
fermentor at the rate of 550 rpm. Initial glucose concentration was
102.9 grams/L. The glucose and succinic acid concentrations were
measured using HPLC techniques. In a parallel experiment,
fermentation was carried out in a total volume of 18,000 ml with 6N
NH.sub.4OH as the neutralizing base. 100 mM NH.sub.4HCO.sub.3 was
provided as the source of inorganic carbon. Additional inorganic
carbon source was provided by supplying carbon dioxide mixed with
1% air (99% CO2/1% air) at the rate of 0.1 vvm. KJ122 biocatalyst
was inoculated at the initial OD.sub.550 nm of 6.1 and the pH was
maintained at 6.75. Fermentation fluid was stirred by operating the
impeller within the fermentor at the rate of 300 rpm. Initial
glucose concentration was 104.7 grams/L. In the fermentation run
supplied only with carbon dioxide, at the end of 36 hours of
fermentation, nearly about 20% of the initially added glucose was
still present while in the fermentation run supplied with carbon
dioxide gas containing 1% air, the glucose consumption was complete
by 36 hour (FIG. 11). In addition, as the result shown in FIG. 12
indicates, the succinic acid productivity was slightly higher in
the microaerated sample when compared to the control sample.
[0093] In the next set of experiments, efforts were made to compare
the effect of different levels of microaeration on fermentation
profile. In these experiments, fermentation was run with 6N
NH.sub.4OH as the neutralizing base. The pH of the fermentation
vessel maintained at 6.75 and the fermentation temperature was kept
at 37.degree. C. Glucose was provided as the source of organic
carbon at the concentration of 100 g/L. 100 mM KHCO.sub.3 was
provided as the source of inorganic carbon. Additional source of
inorganic carbon was provided by supplying carbon dioxide gas
either alone or mixed with definite amount of air. Thus in a
fermentation run with an initial volume of 9,000 ml, aeration was
provided with carbon dioxide gas containing 3% air at the rate of
0.1 vvm and the impeller within the fermentor was operated at 550
rpm. In another fermentation run with an initial volume of 18,000
ml, aeration was provided at the rate of 0.1 vvm with carbon
dioxide gas containing 2% air. The fermentation solution was
stirred by operating the impeller within the fermentor at 300 rpm.
In the third fermentation run with an initial volume of 27,000 ml,
aeration was provided at the rate of 0.037 vvm with carbon dioxide
gas containing 0.5% air. The fermentation fluid was stirred by
operating the impeller within the fermentor at 200 rpm. As the
results shown in FIGS. 13 and 14 indicate excess amounts of oxygen
supply decreased both the titer and the productivity for succinic
acid. Another notable advantage in microaerating the fermentation
vessel was related to byproduct accumulation during succinic acid
fermentation. With microaeration, it was possible to decrease the
amount of byproducts such as pyruvic acid, malic acid, and lactic
acid when compared to the levels of these byproducts in the
fermentation with high oxygen supply (Table 2).
TABLE-US-00001 TABLE 1 Ratio of NH.sub.4OH and NH.sub.4HCO.sub.3
tested to identify an optimal composition for succinic acid
fermentation. Succinic Acetic Succinic Acid Acid Acid Fermentation
NH.sub.4OH NH.sub.4HCO.sub.3 % Molar Titer Titer Yield number (M)
(M) Bicarbonate* (g/L) (g/L) (g/g) 1 5 1 16.7 34.4 2.9 77.72 2 5 2
28.6 76.8 2.8 83.29 3 5 3 37.5 79.0 2.7 83.40 4 6 1 14.3 21.0 1.7
69.61 5 6 2 25 79.4 5.8 79.62 6 6 3 33.3 80.1 3.5 83.51 7 7 1 12.5
21.9 2.3 71.5 8 7 2 22.2 79.6 6.4 80.4 9 7 3 30 81.2 3.4 84.06 10 8
1 11.1 12.8 0.8 65.61 11 8 2 20 60.4 5.4 78.73 12 8 3 27.3 82.9 4.4
81.95 *% Molar Bicarbonate is the percentage of molar concentration
of ammonium bicarbonate in the succinic acid fermentation medium
with reference to the molar concentration of total ammonium
compounds present in the fermentation medium.
TABLE-US-00002 TABLE 2 Succinic acid and other byproducts in 20 L
fermentation runs with different levels of aeration Succinic
Pyruvic Malic Acetic Lactic acid acid acid acid acid Yield
Condition (g/l) (g/l) (g/l) (g/l) (g/l) (g/g) Low O.sub.2 47.6 0
0.6 5.4 0 75.9 (99.5% CO.sub.2 + 0.5% Air) High O.sub.2 44.7 7.3
8.3 4.6 0.4 62.2 (97% CO.sub.2 + 3% Air)
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