Process Biochemistry 41 (2006) 801808

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Kinetics of butyric acid fermentation of glucose and xylose by

Clostridium tyrobutyricum wild type and mutant
Xiaoguang Liu, Shang-Tian Yang *
Department of Chemical Engineering, The Ohio State University, 140 West 19th Avenue, Columbus, OH 43210, USA
Received 9 August 2005; received in revised form 27 September 2005; accepted 3 October 2005

Abstract
The kinetics of butyric acid fermentation by Clostridium tyrobutyricum at pH 6.0 and 37 8C were studied with the wild type ATCC 25755 and its
mutant PPTA-Em, which was obtained from integrational mutagenesis to inactivate the chromosomal pta gene, encoding phosphotransacetylase
(PTA). The potential of using this mutant to improve butyric acid production from glucose and xylose was evaluated in both free and immobilized
cell fermentations. Compared to the wild type, in free cell fermentations PPTA-Em produced 15% more butyrate (0.38 g/g versus 0.33 g/g) from
both glucose and xylose, at much higher concentrations (37.2 g/L versus 22.9 g/L from glucose and 33.5 g/L versus 19.4 g/L from xylose). The
increased butyrate production in the mutant can be attributed to the reduced acetate production as well as reduced specific growth rate. The acetate
yield in the mutant was reduced by 13.5% (0.058 g/g versus 0.067 g/g) and 32% (0.045 g/g versus 0.066 g/g) from glucose and xylose, respectively.
The mutants specific growth rate was reduced by 36% (0.137 h
1 versus 0.214 h
1) on glucose and 26% (0.086 h
1 versus 0.116 h
1) on xylose. A
fibrous-bed bioreactor (FBB) was used to immobilize PPTA-Em mutant cells and further improve butyric acid production. The final butyric acid
concentrations in fed-batch fermentations reached 49.9 g/L from glucose and 51.6 g/L from xylose, with the butyrate yield increased to 0.44 g/g
glucose and 0.45 g/g xylose. As evidenced by the greatly increased butyrate/acetate ratio in the final product profile, it is concluded that the
mutants metabolic pathway has been shifted to favor butyrate production due to the knockout of pta gene even though acetate production remains
at a significant level. The observed metabolic shift is corroborated by the changed protein expression patterns as seen in two-dimensional protein
electrophoresis and SDS-PAGE.
# 2005 Elsevier Ltd. All rights reserved.

Keywords: Clostridium tyrobutyricum; Butyric acid fermentation; Fibrous-bed bioreactor

1. Introduction There has been increasing interest in the production of butyric

Clostridium tyrobutyricum, a gram-positive, rod-shaped,
spore-forming, and obligate anaerobic bacterium, can produce
butyric acid, acetic acid, hydrogen and carbon dioxide from
various carbohydrates including glucose and xylose [1]. Butyric
acid is a short-chain fatty acid naturally generated by anaerobic
fermentation of dietary substrates in intestines. It is currently
produced by petrochemical routes and has many applications; in
the chemical industry, it is mainly used to synthesize butyryl
polymers; in the food industry, it is used to enhance butter-like
notes in food flavors; and in the pharmaceutical industry, it can be
used to treat colorectal cancer and hemoglobinopathies [2,3].
Also, the esters of butyrate are used as additives for increasing
fruit fragrance and as aromatic compounds in perfumes.
* Corresponding author. Tel.: +1 614 292 6611; fax: +1 614 292 3769.
E-mail address: yang.15@osu.edu (S.-T. Yang).
1359-5113/$ see front matter # 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.procbio.2005.10.009
acid from biomass using C. tyrobutyricum and C. butyricum [4
7]. However, the conventional butyric acid fermentation process
is not yet economically competitive because of its low reactor
productivity, low final product concentration, and low product
yield. Recently, C. tyrobutyricum cells immobilized in a fibrous-
bed bioreactor (FBB) were successfully used for butyrate ferm-
entation with increased reactor productivity and final product
concentration [7]. A novel extractive fermentation process has
also been developed, using Alamine 336 in oleyl alcohol as the
extractant contained in a hollow-fiber membrane extractor for
selective removal of butyric acid from the fermentation broth,
that can further enhance product concentration and purity while
lowering the recovery and purification costs [1,8,9].
Creating metabolically engineered mutants of clostridial
strains is another approach for improving fermentation.
Recently, integrational mutagenesis has been successfully used
to create gene knockout mutants [10,11]. In this technique, a
non-replicative integrational plasmid containing a fragment of
802 X. Liu, S.-T. Yang / Process Biochemistry 41 (2006) 801808

the target gene and a selection marker is constructed and then
inserted into the parental chromosome by homologous
recombination to knockout the target gene, resulting in mutants
with altered metabolic pathways and fermentation character-
istics. Using a non-replicative plasmid containing a partial pta
gene, encoding PTA (phosphotransacetylase), Green et al.
obtained mutants of solventogenic C. acetobutylicum ATCC 824
with much reduced PTA and AK (acetate kinase) activities and
acetate production [10]. Similarly, by using the integrational
plasmid pPTA-Em, a pta-deleted C. tyrobutyricum mutant
(PPTA-Em) with decreased PTA activity and increased butyrate
production was obtained [11].
The main objective of this work was to evaluate the potential
of using the metabolically engineered C. tyrobutyricum mutant
PPTA-Em for butyric acid production from glucose and xylose.
In this study, free cell and immobilized cell FBB fermentations
were carried out at 37 8C and pH 6.0 with PPTA-Em and the wild
type for comparison purpose. The effects of integrational
mutagenesis, sugar source, and cell adaptation in the FBB on
butyric acid fermentation kinetics were studied and are discussed
in this paper. Finally, SDS-PAGE and two-dimensional protein
electrophoresis were used to study and characterize protein
expression changes as affected by the mutation in PPTA-Em and
different sugar sources used in the fermentations.
2. Materials and methods
was connected to the 5-L stirred-tank fermentor containing the medium through
a recirculation loop (1 m long, tubing i.d.: 3.1 mm; Microflex Norprene
06402-16, Cole Parmer, Chicago, IL) and operated under well-mixed conditions
with pH and temperature controls. The FBB was first operated at a repeated
batch mode to increase the cell density in the fibrous-bed to a stable, high level
(>50 g/L). To adapt the culture to tolerate a higher butyrate concentration, the
reactor was then operated at fed-batch mode by pulse-feeding a concentrated
substrate solution whenever the sugar level in the fermentation broth was close
to zero. At the end of each fed-batch experiment, the medium in the fermentor
was completely replaced with 2 L of fresh medium to start new fed-batch
fermentation.
2.4. Analytical methods
Cell density was analyzed by measuring the optical density of the cell
suspension at a wavelength of 600 nm (OD600) with a spectrophotometer
(Sequoia-turner, Model 340). One unit of OD600 corresponded to 0.68 g/L of
cell dry weight for cells grown in the glucose medium. A high-performance
liquid chromatography (HPLC) was used to analyze the organic compounds,
including glucose, xylose, lactate, acetate, and butyrate, in the fermentation
broth. The HPLC system consisted of an automatic injector (Shimazu SIL-
10Ai), a pump (Shimadzu LC-10A), organic acid analysis column (Bio-Rad
HPX-87H), a column oven at 45 8C (Shimadzu RID-10A), and a refractive
index detector (Shimadzu RID-10A). The eluent was 0.01N H2SO4, at a flow
rate of 0.6 mL/min. Hydrogen and carbon dioxide production during the
fermentation was monitored using an on-line respirometer (Micro-oxymax
system, Columbus Instrument, Columbus, OH). The fermentor was connected
to a tightly sealed bottle (volume: 5400 mL) for the collection of the gas
products, which was flushed with nitrogen periodically. The change of gas
composition inside the bottle was monitored and used to estimate the gas

2.1. Cultures and medium

C. tyrobutyricum ATCC 25755 was maintained on reinforced clostridial

medium (RCM; Difco) plates in an anaerobic chamber (95% N2, 5% H2).
Working cultures were grown at 37 8C in a previously described synthetic
medium (CGM) with glucose as the substrate [12]. The metabolically engi-
neered mutant, PPTA-Em, was obtained by transforming C. tyrobutyricum
competent cells with non-replicative plasmids containing erythromycin (Em)
resistant gene and partial pta fragment obtained from PCR amplification [11].
The pta knockout mutant, created through homologous recombination of the
plasmids with the chromosome, was selected and stored on the RCM plates
containing 40 mg/mL Em.

2.2. Fermentation kinetic studies

Fed-batch fermentations of C. tyrobutyricum were performed in a 5-L

stirred-tank fermentor (Marubishi MD-300) containing 2 L of the medium
with either glucose or xylose as the substrate. Erythromycin was not used in the
fermentation study as the mutant PPTA-Em was genetically stable without the
selection marker. Anaerobiosis was reached by initially sparging the medium
with nitrogen. The medium pH was adjusted to 6.0 with 6N HCl before
inoculation with 100 mL of cell suspension prepared in a serum bottle.
Experiments were carried out at 37 8C, 150 rpm for agitation, and pH
6.0  0.1 controlled by adding NH4OH. The fed-batch mode was operated
by pulse feeding concentrated substrate solution when the sugar level in the
fermentation broth was close to zero. The feeding was continued until the
fermentation ceased to produce butyrate due to product inhibition. Samples
were taken at regular intervals from the fermentation broth for the analyses of
cell, substrate and acid products.

2.3. Fermentation in fibrous-bed bioreactor

Fig. 1. Kinetics of fed-batch fermentations of glucose by free cells of C.
The fibrous-bed bioreactor (FBB) was made of a glass column packed with tyrobutyricum wild type (A) and mutant PPTA-Em (B) at pH 6.0 and 37 8C:
spiral wound cotton towel and had a working volume of 480 mL. Detailed OD600 (), glucose ( ), butyrate (*), acetate (4), hydrogen (*), and carbon
description of the reactor construction has been given before [12]. The reactor dioxide ( ).
 X. Liu, S.-T. Yang / Process Biochemistry 41 (2006) 801808 803

pellets suspended in 10 mL of 25 mM TrisHCl buffer (pH 7.4) were sonicated,

and the protein extract was collected by centrifugation.
Protein samples for sodium dodecyl sulfate-polyacrylamide gel electro-
phoresis (SDS-PAGE) were prepared from the cell extract after sonication
and centrifugation. The cell extract (10 mL) was concentrated using four
volumes of acetone (40 mL) to precipitate protein at
20 8C overnight, and
re-dissolved in 2 mL of 25 mM TrisHCl buffer (pH 7.4), following the
standard protocol (Bio-Rad, Hercules, CA). Protein samples, 24 mg per well,
were loaded into 12.5% SDS-PAGE gel, and run at 100 V for 2.5 h with
PROTEAN II xi Cell (Bio-Rad) and stained following the instruction of the
manufacturer.
For two-dimensional protein electrophoresis (2DE), cell extract was
concentrated by acetone and then dissolved in the rehydration buffer (8 M
urea, 4% CHAPS, 10 mM DTT, 0.2% (w/v) Bio-Lytes 3/10) for sample
preparation. The first dimension was performed on an 11 cm IPG strip with
a nonlinear immobilized pH 310 gradient (Amersham, Piscataway, NJ). The
IPG strip was rehydrated in the rehydration buffer with 20 mg protein sample at
50 V for 12 h using PROTEAN IEF Cell (Bio-Rad). After rehydration, the
protein was focused on IPG strip by preset method, at 250 V for 15 min to
remove excess salts, then ramped linearly from 250 to 5000 V for 2 h, and
finally maintained at 8000 V for 4.5 h for focusing purpose. After isoelectric
focusing (IEF), the strip was equilibrated in equilibrate buffer I (6 M urea, 2%
SDS, 0.375 M TrisHCl, pH 8.8, 20% glycerol and 130 mM DTT) for 10
15 min and in equilibrate buffer II (6 M urea, 2% SDS, 0.375 M TrisHCl, pH
8.8, 20% glycerol and 135 mM iodoacetamide) for 1015 min. The equili-
brated strip was applied to a polyacrylamide/PDA SDS gel to run the second
dimension electrophoresis at 100 V for 120180 min with Mini-PROTEAN 3
Cell (Bio-Rad). The protein spots were developed using silver staining kit
(Amersham). The two-dimensional protein electrophoresis maps were ana-
lyzed by using Phoretix 2D software (Nonlinear Dynamics Ltd, Newcastle
upon Tyne, UK).

Fig. 2. Kinetics of fed-batch fermentations of xylose by free cells of C.

tyrobutyricum wild type (A) and mutant PPTA-Em (B) at pH 6.0 and 37 8C: 3. Results and discussion
OD600 (), glucose ( ), butyrate (*), acetate (4), hydrogen (*), and carbon
dioxide ( ). 3.1. Fermentation kinetics

production, which was also measured by collecting the produced gas in an
inverted 4-L plastic graduate cylinder in a water trough.
2.5. Preparation of cell extract and protein electrophoresis
Cells cultivated in 100 mL of CGM at 37 8C were allowed to grow to the
exponential phase (OD600 = 1.5), and then harvested and washed. The cell
Typical fed-batch fermentation kinetics with C. tyrobutyr-
icum wild type and PPTA-Em mutant grown on glucose and
xylose are shown in Figs. 1 and 2, respectively. In general, there
was a short lag phase with little gas (H2 and CO2) and acid
(acetic and butyric acids) production, which then increased
during the exponential phase. For the wild type, acid production
either stopped or slowed down dramatically when cells entered

Table 1
Comparison of fermentations of glucose by C. tyrobutyricum wild type and PPTA-Em at 37 8C, pH 6.0
Wild type PPTA-Em
Free cell Free cell Immobilized cell
Cell growth
Specific growth rate (h
1) 0.214  0.044 0.137  0.032 0.095  0.036
Biomass yield (g/g) 0.109  0.019 0.141  0.024 0.087  0.008
Acid production
Butyric acid concentration (g/L) 22.90  4.06 37.22  4.80 49.85  0.51
Butyric acid yield (g/g) 0.33  0.01 0.38  0.01 0.44  0.01
Acetic acid concentration (g/L) 4.15  0.59 4.19  0.013 8.74  0.63
Acetic acid yield (g/g) 0.067  0.012 0.058  0.004 0.081  0.005
Butyrate/acetate ratio (g/g) 5.52 8.88 5.70
Gas production
H2 yield (g/g) 0.017  0.002 0.018  0.001 0.016  0.001
CO2 yield (g/g) 0.360  0.035 0.389  0.027 0.388  0.005
H2/CO2 ratio (mol/mol) 1.05  0.03 1.05  0.03 0.93  0.02
Note: average  standard deviation were calculated from two (wild type) or three (mutant) batch fermentations.
804 X. Liu, S.-T. Yang / Process Biochemistry 41 (2006) 801808

Table 2
Comparison of fermentations of xylose by C. tyrobutyricum wild type and PPTA-Em at 37 8C, pH 6.0
Wild type PPTA-Em
Free cell Free cell Immobilized cell
Cell growth
Specific growth rate (h-1) 0.116  0.009 0.086  0.020 0.048  0.006
Biomass yield (g/g) 0.095  0.003 0.109  0.013 0.069  0.003
Acid production
Butyric acid concentration (g/L) 19.42  1.195 33.49  2.89 51.55  3.49
Butyric acid yield (g/g) 0.33  0.02 0.38  0.02 0.45  0.02
Acetic acid concentration (g/L) 3.31  0.01 3.89  0.26 6.42  1.60
Acetic acid yield (g/g) 0.066  0.006 0.045  0.001 0.045  0.016
Butyrate/acetate ratio (g/g) 5.87 8.61 8.02
Gas production
H2 yield (g/g) 0.017  0.001 0.017  0.001 0.015  0.001
CO2 yield (g/g) 0.365  0.001 0.373  0.031 0.348  0.004
H2/CO2 ratio (mol/mol) 1.07  0.02 1.05  0.02 0.96  0.01
Note: average  standard deviation were calculated from two (wild type) or three (mutant) batch fermentations.

the stationary phase, although gas production and glucose (or biomass yield with xylose as the substrate is because that xylose
xylose) consumption continued for a substantial period. For the fermentation gives lower energy efficiency as compared with
PPTA-Em mutant, acetic acid production also stopped in the glucose fermentation. The different specific growth rate and
stationary phase; however, butyric acid production continued to biomass yield from the mutant suggest that the carbon and
reach a much higher level. Consequently, the mutant produced energy fluxes have been redistributed in the metabolic pathways
much more butyric acid and reached a higher final butyric acid of the mutant, which also led to the significant changes in acids
concentration when the fed-batch fermentations were finally production.
stopped. The fermentations were stopped when the sugar
substrate was no longer consumed by the cells due to inhibition
by butyric acid [10].
It is clear that PPTA-Em is more tolerant to butyric acid
inhibition, a result from the genetic manipulation of the pta
gene in the mutant [11]. The specific growth rates and product
yields from glucose and xylose in these fermentations were
estimated and are listed in Tables 1 and 2, respectively. The
specific growth rate was determined from the slope of the semi-
logarithmic plot of OD versus time using the time-course data
in the exponential phase. The product yield was determined
from the linear plot of product concentrations versus substrate
concentrations at various fermentation times during the batch
fermentation. The effects of the mutation on C. tyrobutyricum
and the butyric acid fermentation are further discussed in the
following sections.

3.2. Effect on cell growth

The mutants specific growth rate was reduced by 36%

(0.137 h
1 versus 0.214 h
1) on glucose (Table 1) and 26%
(0.086 h
1 versus 0.116 h
1) on xylose (Table 2), respectively.
However, the cell biomass yields for the mutant appeared to be
higher than those for the wild type, although the difference was
within the error range. The lower specific growth rate for the
mutant can be attributed to the metabolic burden on cells caused
by possibly less energy (ATP) generation in the sugar Fig. 3. SDS-PAGE of cellular proteins from C. tyrobutyricum. (Lane 1: wild
type, free cell with glucose; Lane 2: wild type, free cell with xylose; Lane 3:
metabolism due to pta knockout. This is mainly because that PPTA-Em, free cell with glucose; Lane 4: PPTA-Em, free cell with xylose; Lane
pyruvate oxidation to acetate can generate more ATP than its 5: PPTA-Em, immobilized cell with glucose; Lane 6: PPTA-Em, immobilized
oxidation to butyrate. The lower specific growth rate and cell with xylose; Lane 7: molecular size marker.)
 X. Liu, S.-T. Yang / Process Biochemistry 41 (2006) 801808 805

3.3. Effects on butyric and acetic acids production
Compared to the wild type, the PPTA-Em mutant not only
can tolerate and produce a much higher concentration of butyric
acid, it also gives higher butyric acid yields from glucose and
xylose. As can be seen in Tables 1 and 2, PPTA-Em produced
15% more butyrate (0.38 g/g versus 0.33 g/g) from both
glucose and xylsoe. The increased butyrate production in the
mutant can be attributed to the reduced acetate production as
well as reduced specific growth rate. The acetate yield in the
mutant was reduced by 13.5% (0.058 g/g versus 0.067 g/g)
from glucose and 32% (0.045 g/g versus 0.066 g/g) from
xylose, respectively. Although the mutant still produced a
significant amount of acetate, the inactivation of pta gene
reduced acetic acid and increased butyric acid production
because more substrates were directed toward the butyric acid
formation pathway, as evidenced by the increased butyrate/
acetate (B/A) ratio (from 5.5 to 8.9 g/g) in the fermenta-
tions. This metabolic shift was the direct result of pta knockout
mutation.
The final butyric acid concentrations produced from glucose
and xylose by PPTA-Em increased by 62% (from 22.9 to
37.2 g/L) and 72% (from 19.4 to 33.5 g/L), respectively. Our
previous study has shown that the acetic acid-forming enzymes
(PTA and AK) are more sensitive to butyric acid inhibition than
butyric acid-forming enzymes (phosphotransbutyrylase and
butyrate kinase) [13]. The mutants reduced sensitivity to
butyric acid inhibition thus can be attributed to the reduced
carbon flux through the PTAAK pathway. However, the final
acetic acid concentration produced in the fermentation by

Fig. 4. Two-dimensional protein electrophoresis for wild type (A) and mutant (B) grown on glucose.
806 X. Liu, S.-T. Yang / Process Biochemistry 41 (2006) 801808

PPTA-Em was not much affected even though the acetic acid
yield from sugar was reduced significantly. This is because that
there may be other enzymes or pathways present in C.
tyrobutyricum that can also produce acetate from pyruvate or
acetyl CoA [11].
3.4. Effect on gas production
As discussed before, both hydrogen and carbon dioxide were
produced throughout the fermentation. The pta knockout
mutation did not appear to have any significant effect on gas
production by the PPTA-Em mutant. The average hydrogen and
carbon dioxide yields were found to be 0.017 and 0.37 g/g,
respectively, for both the mutant and the wild type using either
glucose or xylose as the substrate.
3.5. SDS-PAGE and 2-DE analyses of protein expression
The effects of pta knockout and fermentation conditions on
protein expression were first studied with SDS-PAGE. As can
be seen in Fig. 3, there are notable differences in the SDS-PAGE
protein profiles obtained from the wild type and the mutant. For
example, the level for the proteins with molecular weight (MW)
of 32 kDa was significantly lowered for PPTA-Em mutant
grown on glucose. Interestingly, this group of 32 kDa proteins
in the mutant was much higher when xylose was used as the
growth substrate. This substrate effect, however, was not
observed with the wild type. It is clear that a single gene
knockout can induce rather complicated responses in protein
expression by the cells due to gene and metabolic regulatory
networks.

Fig. 5. Two-dimensional protein electrophoresis for wild type (A) and mutant (B) grown on xylose.
 X. Liu, S.-T. Yang / Process Biochemistry 41 (2006) 801808 807

Table 3
Comparison of protein expression in the wild type and mutant of C. tyrobutyricum using either glucose or xylose as the carbon source
Protein Glucose Xylose
PI MW (kDa) Wild type PPTA-Em Wild type PPTA-Em
N volume (spot #) R volume (%) R volume (%) (spot #) R volume (%) (spot #) R volume (%) (spot #)
5.89 35.51 0.166 (87) 100 221.7 (86) 0 0
5.99 34.26 0.090 (88) 100 482.2 (87) 157.8 (100) 211.1 (98)
6.17 34.02 0.261 (90) 100 167.4 (88) 386.6 (101) 243.7 (99)
6.32 31.10 0.302 (89) 100 0 184.4 (105) 42.7 (102)
6.49 33.86 0.727 (92) 100 77.0 (91) 139.3 (106) 132.7 (100)
6.65 34.02 0.347 (91) 100 34.0 (90) 213.8 (107) 24.8 (105)
6.77 33.63 0.343 (93) 100 0 147.8 (103) 0
6.90 33.55 0.802 (95) 100 32.3 (92) 52.1 (104) 38.9 (103)
7.19 33.63 0.051 (94) 100 27.5 (89) 0 0
Notes: (1) the protein spot number in the parenthesis is the same number labeled on the two-dimensional protein electrophoresis gels shown in Figs. 3 and 4. (2) The
expression data reported here are the average values of two gels. (3) N volume: normalized spot volume on the 2DE gel; R volume: relative spot volume to the wild
type grown on glucose as the reference.

The effects on protein expression were further studied with both glucose and xylose can be efficiently used in butyric acid
two-dimensional protein electrophoresis (2DE). There were fermentation and there is no obvious preference for either
200 protein spots on the 2DE maps obtained for both the wild glucose or xylose as the carbon source, although cell growth
type and mutant PPTA-Em grown on glucose (Fig. 4). A smaller and biomass yield from xylose were significantly lower than
number of protein spots were obtained for growth on xylose those from glucose. The lower growth rate from xylose should
(Fig. 5). These 2DE protein maps were analyzed with the not be a concern for immobilized cell fermentation using the
Phoretix 2D AdvancedTM software, which compared and FBB, which is discussed below.
identified proteins with changed expression levels after normal-
izing the different intensities of the protein spots on these 2DE
gels. As expected, the number of protein spots and their
intensities (expression levels) on the 2DE gels were different
between the wild type and the mutant. For example, in the region
circled on the gels (PI: 5.57.5, MW: 32 kDa) there are two
more protein spots for the wild type than for the mutant. These
missing protein spots are very likely to include PTA and AK, but
cannot be identified in this work due to lack of proteomic
information for C. tyrobutyricum. Table 3 shows the results of
2DE analysis of proteins in this region. The expression levels of
these protein spots with different PIs and MWs are compared
based on the normalized protein expression volume. With the
wild type grown on glucose as the reference, above 100%
indicates up regulation while below 100% indicates down
regulation of the protein expression in the mutant or growth on
xylose. For growth on glucose, two proteins (spot #89, PI: 6.3,
MW: 31.1; spot #93, PI: 6.77, MW: 33.6) found in the wild type
were missing and at least one protein (spot #95, PI: 6.90 and MW:
33.55) highly expressed in the wild type (N volume = 0.802) was
dramatically down-regulated in the mutant. Similarly, for growth
on xylose, the protein spot #93 (PI: 6.77, MW: 33.6) found in the
wild type was also missing in the mutant. It is noted that protein
expression is also significantly affected by the carbon source used
in the fermentation. However, the pta knockout has similar
effects on protein expression and metabolic pathway shift,
regardless of the carbon source used in the fermentation.

3.6. Effects of carbon source

Fig. 6. Kinetics of fed-batch fermentations by C. tyrobutyricum mutant PPTA-
Em immobilized in the FBB with glucose (A) and xylose (B) as the substrate at
Clostridium tyrobutyricum is one of a few bacteria that can pH 6.0 and 37 8C: OD600 (), glucose ( ), butyrate (*), acetate (4), hydrogen
use xylose to produce butyric acid. As observed in this work, (*), and carbon dioxide ( ).
808 X. Liu, S.-T. Yang / Process Biochemistry 41 (2006) 801808
3.7. Immobilized cell fermentation in FBB
Immobilized cell bioreactors have the potential in improving a
fermentation process by increasing cell density, reactor
productivity and final product concentration. We have previously
developed a fibrous-bed bioreactor (FBB) for immobilized cell
fermentations to produce several organic acids from biomass
with significantly improved productivity, yield, and final product
concentration [1214]. The FBB was thus applied to the PPTA-
Em mutant in this work to further improve butyric acid
production from glucose and xylose. As shown in Fig. 6, the FBB
fermentations produced much more butyric acid from glucose
and xylose than those obtained in free cell fermentations. As cells
adapted in the FBB became more tolerant to butyrate inhibition,
they were able to produce butyric acid at a final concentration of
50 g/L with a butyrate yield of 0.45 g/g sugar (see Tables 1
and 2). The improvements over the free cell fermentations were
over 16.5% in butyrate yield and 3454% in the final butyrate
concentration. The improved butyrate yield can be attributed to
the reduced cell growth in the immobilized cell fermentation,
whereas the improved butyrate tolerance is the result of
adaptation and possibly physiology changes in the FBB
environment [13,15]. SDS-PAGE protein expression profiles
(see Fig. 3) for cells from the FBB showed similar patterns to
those in the free cell fermentations, however. Further study and
characterization of cells adapted in the FBB will be necessary in
order to fully understand why the FBB fermentation allowed the
cells to tolerate and produce more butyric acid.
4. Conclusions
The potential of using the C. tyrobutyricum mutant obtained
from integrational mutagenesis that selectively inactivated the
chromosomal pta gene, to produce butyrate from glucose and
xylose was studied. Compared with the wild type, butyric acid
production from glucose and xylose by this mutant is improved
with higher butyrate yields, final product concentrations, and
product purity (B/A ratio). The butyric acid fermentation was
further improved by immobilizing the mutant in the fibrous-bed
bioreactor to facilitate cell adaptation to attain even higher
product yields and concentrations. Since both glucose and
xylose, which are rich in corn fiber hydrolysate [16], can be
used equally well to produce butyric acid, it is feasible to
produce butyric acid from mixed sugars derived from abundant
plant biomass such as corn fiber. As demonstrated in this work,
the butyric acid fermentation can be more efficiently carried out
in the FBB using the metabolically engineered mutant of C.
tyrobutyricum, allowing economical production of bio-based
butyric acid.
Acknowledgements
This work was supported in part by research grants from the
Department of Energy (DE-FG02-00ER86106), the U.S.
Department of Agriculture (CSREES 99-35504-7800), and
the Consortium for Plant Biotechnology Research Inc. (CPBR;
R-82947901).
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