-
Accepted Manuscript
Propionic acid production in glycerol/glucose co-fermentation by
Propionibac
terium freudenreichii subsp. shermanii
Zhongqiang Wang, Shang-Tian Yang
PII: S0960-8524(13)00367-2
DOI: http://dx.doi.org/10.1016/j.biortech.2013.03.012
Reference: BITE 11471
To appear in: Bioresource Technology
Received Date: 14 January 2013
Revised Date: 2 March 2013
Accepted Date: 4 March 2013
Please cite this article as: Wang, Z., Yang, S-T., Propionic
acid production in glycerol/glucose co-fermentation by
Propionibacterium freudenreichii subsp. shermanii, Bioresource
Technology (2013), doi: http://dx.doi.org/10.1016/
j.biortech.2013.03.012
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1
Propionic acid production in glycerol/glucose co-fermentation
by
Propionibacterium freudenreichii subsp. shermanii
Zhongqiang Wang and Shang-Tian Yang*
William G. Lowrie Department of Chemical &Biomolecular
Engineering, The Ohio State
University, 140 W 19th Ave, Columbus, OH 43210, USA
*Corresponding author: phone: +614 292 6611; fax: +614 292 3769;
email: [email protected]
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Abstract
Propionibacterium freudenreichii subsp. shermanii can ferment
glucose and glycerol to
propionic acid with acetic and succinic acids as two
by-products. Propionic acid production from
glucose was relatively fast (0.19 g/Lh) but gave low product
yield (~0.39 g/g) and selectivity
(P/A: ~2.6; P/S: ~4.8). In contrast, glycerol with a more
reduced state gave a high propionic acid
yield (~0.65 g/g) and selectivity (P/A: ~31; P/S: ~11) but low
productivity (0.11 g/Lh). On the
other hand, co-fermentation of glycerol and glucose at an
appropriate mass ratio gave both a high
yield (0.540.65 g/g) and productivity (0.180.23 g/Lh) with high
product selectivity (P/A:
~14; P/S: ~10). The carbon flux distributions in the
co-fermentation as affected by the ratio of
glycerol/glucose were investigated. Finally, co-fermentation
with cassava bagasse hydrolysate
and crude glycerol in a fibrous-bed bioreactor was demonstrated,
providing an efficient way for
economic production of bio-based propionic acid.
Keywords: Propionibacterium freudenreichii subsp. shermanii;
Propionic acid; Glycerol; Co-
fermentation; Fibrous-bed bioreactor
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1. Introduction
Propionic acid is a C3 carboxylic acid with many industrial
applications as a specialty chemical
and its calcium, potassium and sodium salts are widely used as
food and feed preservatives
(Boyaval and Corre, 1995). Currently, propionic acid is produced
almost exclusively via
petrochemical processes, with an annual production capacity of
~400 million lbs in the US. As
the crude oil prices had surpassed US$100 per barrel, there have
been increasing interests in
propionic acid production from renewable bioresources by
fermentation using propionibacteria
(Feng et al., 2010; Goswami and Srivastava, 2001; Jin and Yang,
1998; Martnez-Campos and de
la Torre, 2002; Paik and Glatz, 1994; Rickert et al., 1998;
Suwannakham et al., 2006; Wang et
al., 2012; Zhang and Yang, 2009ab; Zhu et al., 2012), a group of
gram-positive, facultative
anaerobic, non-spore forming bacteria that have long been used
in the production of Swiss-type
cheese and vitamin B12 (Thierry et al., 2011) and also recently
recognized for their probiotic
properties for human consumption. However, conventional
propionic acid fermentation suffers
from low productivity and yield due to strong end-product
inhibition and the co-production of
other byproducts, mainly acetic and succinic acids. To lower the
product cost, recent efforts have
focused on using industrial wastes or byproducts as low-cost
renewable feedstocks for propionic
acid fermentation (Feng et al., 2011; Liang et al., 2012; Zhu et
al., 2012).
With the fast growth of biodiesel production, a promising
substitute for petroleum diesel,
a large amount of crude glycerol, about 10% (w/w) of the
biodiesel produced, is generated
annually, making crude glycerol an economically feasible
feedstock for industrial uses (da Silva
et al., 2009). Several studies have shown that glycerol can be a
good carbon source for propionic
acid fermentation with a higher propionic acid yield and much
lower acetic acid formation
compared to glucose (Barbirato et al., 1997; Coral et al., 2008;
Himmi et al., 2000; Ruhal and
Choudhury, 2012; Zhang and Yang, 2009b; Zhu et al., 2010).
Glycerol has a high reduction
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degree, which favors the production of more reduced metabolites
(Ito et al., 2005; Malaviya et al.,
2012; Zeng and Biebl, 2002) but can cause redox imbalance in
metabolism, leading to reduced
cell growth and productivity, when used as the sole carbon
source in fermentation (Himmi et al.,
2000; Zhang and Yang, 2009b). To overcome this problem,
co-fermentation of glycerol with
glucose has been proposed as an efficient process supporting
both product formation and cell
growth (Chen et al., 20; Liu et al., 2011; Xiu et al.,
2007).
The goal of this study was to evaluate the feasibility of
producing propionic acid from
crude glycerol present in biodiesel waste and glucose derived
from cassava bagasse in a co-
fermentation process with Propionibacterium freudenreichii
subsp. shermanii. Cassava is an
important food crop in many Asian and Latin American countries
with an annual production of
more than 250 million tons in 2011. Industrial processing of
cassava tuber for starch extraction
yielded significant amounts of bagasse, which was usually used
as animal feed or disposed into
landfills, imposing serious environmental concerns (Pandey et
al., 2000). Bioconversion of
cassava bagasse has previously been studied for the production
of fumaric and lactic acids (Carta
et al., 1999; Thongchul et al., 2009), but never for propionic
acid. In this study, co-fermentation
of cassava bagasse hydrolysate and crude glycerol supplemented
with corn steep liquor in a
fibrous-bed bioreactor (FBB) was demonstrated as an efficient
way for economic production of
bio-based propionic acid. The effects of the glycerol/glucose
mass ratio on NADH availability
and carbon flux distributions in the co-fermentation were also
investigated and are reported in
this paper. This is the first report about the glycerol/glucose
co-fermentation behavior of P.
freudenreichii subsp. shermanii, which offers an environmentally
friendly and sustainable route
for propionic acid production with high product yield and
productivity.
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2. Materials and methods
2.1 Culture and media
The stock culture of P. freudenreichii subsp. shermanii DSM 4902
(DSMZ, Germany) was
cultivated anaerobically at 32C in NLB medium containing (per
liter) 10 g yeast extract, 10 g
trypticase soy broth, and 10 g sodium lactate, in serum tubes
and stored at 4 C. Unless
otherwise noted, fermentation kinetics was studied in a
synthetic medium containing (per liter)
10 g yeast extract, 5 g trypticase soy broth, 0.25 g K2HPO4,
0.05g MnSO4, 20 g CaCO3, and 30 g
carbon source (glucose, glycerol or glycerol/glucose mixture).
All media were sparged with
nitrogen gas, sealed in serum tubes or bottles, and autoclaved
at 121 C for 30 min.
2.2 Preparation of cassava bagasse hydrolysate and crude
glycerol as carbon sources
Cassava bagasse (CB), which contained about 43% starch, 25%
cellulose, 10% hemicellulose
and 10% lignin on a dry weight basis, was obtained from a
cassava-processing factory in
Guangdong, China and was dried and milled to fine powder of
50100 m in diameter. To
prepare the CB hydrolysate, 100 g CB powder mixed with 900 ml
distilled water in a 2-L flask
were autoclaved at 121 C for 30 min. Then, commercial
glucoamylase (Distillase L-400,
activity: 350 GAU/g, Genencor, NY) at a loading of 0.06 g/g CB
(on a dry solids basis) and
cellulase (Accellerase1500, endoglucanase activity: 22002800 CMC
U/g, -glucosidase activity:
525775 pNPG U/g, Genencor, NY) at 0.1 ml/g CB (on a dry solids
basis) were aseptically
added into the flask to hydrolyze starch and cellulose,
respectively, at 58 C, pH 4.3, 200 rpm for
48 h. HCl was used to adjust pH before enzymatic hydrolysis.
After the enzyme treatments, the
hydrolysate was centrifuged at 8,000 rpm for 10 min to remove
insolubles and the supernatant
was stored at 4 C for future use. The CB hydrolysate contained
35.66 g/L glucose, 0.96 g/L
xylose and trace amounts of arabinose and acetic acid.
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6
Crude glycerol present in biodiesel wastewater from a biodiesel
manufacturing plant was
prepared together with corn steep liquor (CSL), as nitrogen
source, from a corn wet-milling plant.
Approximately 90 g crude glycerol solution containing ~40 g
glycerol and 60 g CSL were mixed
in distilled water to a final volume of 500 ml. The pH of this
mixture was adjusted to 6.8 with
ammonium hydroxide. After centrifugation at 8000 rpm for 15 min,
the aqueous phase between a
layer of fatty acids on the top and precipitates on the bottom
was collected and autoclaved at 121
C for 30 min. This sterile solution was then aseptically added
to the bioreactor containing CB
hydrolysate for fermentation kinetics study described later. In
addition to crude protein, amino
acids and trace elements (metal ions and vitamins), CSL used in
this study also contained 0.077
g/g lactic acid as additional carbon source and trace amount of
acetic acid and xylose.
2.3 Batch fermentation
Batch fermentations with glucose, glycerol, and glycerol/glucose
mixture, respectively, as carbon
sources were studied in 125-ml serum bottles and 5-L
bioreactors. Each serum bottle containing
50 ml of the medium was inoculated with 2.5 ml of a freshly
prepared seed culture (OD600 ~3.0)
in NLB medium in a serum tube. The serum bottle cultures were
incubated at 32 oC with pH
buffered with 20 g/L CaCO3, (initial pH 6.8) and samples were
withdrawn periodically with 1-ml
syringes. After centrifugation, clear broth samples were frozen
at -20 C for future analysis.
Unless otherwise noted, duplicate bottles were used for each
condition studied.
Batch fermentations were also carried out in a 5-L stirred-tank
fermentor controlled at 32
C, pH 6.5 by adding 6 N NaOH, and agitation at 50 rpm. The
fermentor containing ~900 ml of
the basic medium without the carbon source and a concentrated
substrate (glucose, glycerol, or
glycerol/glucose mixture) solution (~50 ml) in a flask were
autoclaved at 121 C for 30 min
separately and then mixed aseptically in the fermentor. After
sparging with N2 for 45 min to
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anaerobiosis, the fermentor was inoculated with 50 ml of an
overnight culture (OD600 ~2.0).
Samples were withdrawn at regular time intervals to monitor cell
growth and fermentation
kinetics.
2.4 Repeated batch fermentations in a fibrous-bed bioreactor
Repeated batch fermentations were studied in a 350-ml
fibrous-bed bioreactor (FBB) connected
with a recirculation loop to a 5-L stirred-tank fermentor for
temperature and pH controls. The
FBB was made of a glass column packed with a spirally wound
cotton cloth laminated with a
corrugated stainless steel wire mesh. Detailed description of
the FBB system can be found
elsewhere (Suwannakham and Yang, 2005). After 2448 h incubation,
the fermentation broth
with cells in the 5-L fermentor was recirculated through the FBB
for cell immobilization in the
fibrous bed for 2436 h until the cell density in the broth no
longer decreased. The old broth
was then drained and replaced with a fresh medium to allow the
cells in the FBB to continue to
grow. This process was repeated several times to obtain a stable
and high cell density in the
reactor system. Then, the fermentation kinetics with
glycerol/glucose at a mass ratio of 2 was
studied with three consecutive batches, followed with a batch
with crude glycerol and CB
hydrolysate as substrates. The total liquid volume in each batch
was ~1.5 L, including ~350 ml in
the FBB.
2.5 Stoichiometric analysis of carbon flux distribution
Carbon flux distributions among various metabolites and cell
biomass in the metabolic pathway
(see Fig. S1 in Supplemental Materials) of propionibacteria were
analyzed using a stoichiometric
model (see Table 1) and batch fermentation kinetics data from
5-L bioreactor. The model
involves 6 reactions for glucose fermentation, 5 reactions for
glycerol fermentation and 7
reactions for co-fermentation. The metabolic fluxes were
determined based on several
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assumptions. First, pseudo-steady-state hypothesis was applied
to the intermediate metabolite,
pyruvate. The net formation rate of pyruvate was set to zero so
that there was no accumulation
during fermentation. Second, the system was NADH balanced so
that the production and
consumption rates of this reducing co-factor were equal. Third,
the system was energy sufficient
so that ATP produced in the oxidation of carbon sources and
acetate pathway could meet the
needs of metabolite and biomass synthesis. The carbon flux
distributions were estimated based
on the experimental data on substrates (glucose and glycerol)
consumption and metabolites
production, and the fluxes at the pyruvate node were normalized
to show the mole percentage of
pyruvate formed or consumed in each branch pathway.
2.6 Analytical methods
Cell growth was monitored by measuring the optical density (OD)
at 600 nm in a 1.5-ml cuvette
using a spectrophotometer (Shimadzu, UV-16-1). Broth samples
with suspended cells were
diluted to an OD reading of less than 0.8 with distilled water.
Glycerol, glucose, and organic
acids (acetic, succinic, and propionic acids) were quantified by
using high performance liquid
chromatography (Shimadzu) with an organic acid analysis column
(HPX-87H, Bio-Rad)
operated at 45C with 0.005 M H2SO4 as the mobile phase at 0.6
ml/min.
3. Results and discussion
3.1 Glucose and glycerol fermentations
Batch fermentation kinetics with glucose and glycerol as carbon
source, respectively, were
studied in serum bottles and 5-l bioreactors. Figure 1 shows
batch fermentation kinetics of
glucose and glycerol in bioreactor with pH controlled at 6.5. In
general, the fermentation was
faster with glucose than with glycerol as the substrate, but
more propionic acid was produced
from glycerol on the same weight basis. Theoretically, one mol
glucose produces 4/3 mol
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propionic acid and 2/3 mol acetic acid via the EMP pathway, as
shown in the following equation
(Playne, 1985).
1.5 Glucose 2 Propionic acid + Acetic acid + CO2 + H2O
So the theoretical yield for propionic acid production from
glucose is 0.55 g/g. In contrast, one
mol glycerol produces one mol propionic acid and no acetic acid
via the EMP pathway, with a
theoretical propionic acid yield of 0.80 g/g.
Glycerol Propionic acid + H2O
However, the actual propionic acid yield could be lower due to a
fraction of the substrate
carbon was used for cell biomass or higher if the HMP pathway
was used in glycolysis, which
was affected by the growth conditions. As expected, glycerol
fermentation gave a higher
propionic acid yield than that in glucose fermentation (see
Tables 2 and 3). Compared to glucose,
glycerol with a more reductive state gave a much higher
propionic acid/acetic acid (P/A) product
ratio for balancing the intracellular NADH/NAD+. Glycerol also
gave a higher propionic
acid/succinic acid (P/S) ratio than that in glucose fermentation
in the bioreactor at pH 6.5, but the
P/S ratio was lower in serum bottles without pH control (pH
dropped from 6.8 to 4.8) because of
stronger propionic acid inhibition at the lower pH resulting in
more succinic acid accumulation.
It is noted that the propionic acid yield from glucose in serum
bottles was higher than that in the
bioreactor because cell growth was inhibited in serum bottles,
due to the lower pH, and thus
more substrate carbon was converted to propionic acid. However,
as a result of the lower cell
biomass and pH, the propionic acid productivity was also lower
in serum bottles.
For cells grown in the bioreactor at pH 6.5, the specific growth
rate was unexpectedly higher
in glycerol fermentation than in glucose fermentation although
the final cell density (OD) was
lower with glycerol as carbon source. Clearly, P. shermanii can
use glycerol to support good cell
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growth. In contrast, P. acidipropionici ATCC 4875 did not grow
well with glycerol as the sole
carbon source. When 40 g/l glycerol was used as carbon source,
glycerol could not be
completely used by P. acidipropionici after an extended
culturing period and the final OD was
only 3, much lower than that in glucose fermentation (Zhang,
2009). Nevertheless, compared to
glucose, propionic acid productivity with glycerol as sole
carbon source for P. shermanii was
still low even though glycerol could support good cell growth
with high propionic acid yield and
P/A ratio.
3.2 Co-fermentation of glycerol and glucose
Co-fermentation of glycerol and glucose at various mass ratios
of 1, 2, 3, 4 and 5 was first
investigated in serum bottles and the results are summarized and
compared in Table 2. In general,
increasing the glycerol/glucose mass ratio also increased the
ratio of glycerol consumption rate
to glucose consumption rate from ~1.0 to 1.9, suggesting that
glycerol became increasingly a
more favorable substrate than glucose in the co-fermentation,
which also increased the propionic
acid yield to reach the maximum value of ~0.65 g/g. However, the
P/A ratio remained relatively
stable at ~6, which was more than 2-fold of that in glucose
fermentation but lower than that with
glycerol as sole carbon source (9.6). Clearly, glucose as the
co-substrate allowed a significant
amount of pyruvate to be converted to acetate, which generated
more ATP and resulted in faster
fermentation while still maintained a high propionic acid yield.
Meanwhile, the P/S ratio also
increased to 710, which was comparable to that with glucose
(9.2) and much higher than that
with glycerol (3.1) as sole carbon source.
Since the ratio of glycerol consumption rate to glucose
consumption rate obtained in
serum bottles was between 1 and ~1.9, the glycerol/glucose mass
ratios of 1, 1.5, 2, and 3 in the
co-fermentation were further studied in 5-L bioreactors at pH
6.5. In general, glycerol was
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consumed faster than glucose at all mass ratios studied (Figure
2A; also see Fig. S2 in
Supplemental Materials). However, the fermentation became
significantly slower when the
glycerol/glucose mass ratio increased to 3, although the
specific growth rate was not significantly
different at all conditions studied (see Table 3). The mass
ratio of 2 gave the highest propionic
acid productivity of ~0.23 g/Lh, which was higher than that with
glucose (0.19 g/ Lh) and about
double of that with glycerol (0.11 g/Lh) as sole carbon source.
The propionic acid yield
(0.540.65 g/g) in the co-fermentation was much higher than that
of glucose fermentation (0.39
g/g) but lower than that of glycerol fermentation (0.65 g/g),
except at the higher mass ratio of 3.
Also, both P/A and P/S ratios in the co-fermentation were much
higher than those in the glucose
fermentation. It is thus clear that the fermentation with
glycerol and glucose as co-substrates was
advantageous for propionic acid production.
Batch fermentation was then studied with crude glycerol, CB
hydrolysate and CSL as
low-cost carbon and nitrogen sources, with glycerol/(glucose +
lactate) mass ratio of ~2. Lactic
acid, which has the same reductance degree as glucose, was
present in CSL and also used as
carbon source by propionibacteria. The results are shown in
Figure 2B. In general, the
fermentation kinetics was similar to that with 2
glycerol/glucose in the synthetic medium, with
slightly higher propionic acid yield and productivity and lower
P/S and P/A ratios. The results
showed that the propionibacteria used these inexpensive
feedstocks as efficiently as the more
expensive pure glycerol, glucose, yeast extract and trypticase
for propionic acid production. The
results also suggested no significant inhibition from impurities
present in the crude glycerol as
most of the fatty acids and methanol should have been removed
during media preparation.
3.3 Effects of co-fermentation on carbon flux distributions
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Propionibacteria use the dicarboxylic acid pathway (see Fig. S1
in Supplemental Materials), in
which the substrate or carbon source is first oxidized to
pyruvate via. NADH-generating
glycolysis pathways. Carbon source with a lower oxidation state,
such as glycerol, can generate
more NADH for the same amount of pyruvate produced. From
pyruvate, two mol NADH are
oxidized with the formation of one mol propionic acid, while one
mol NADH is produced with
the synthesis of one mol acetic acid. Partitioning carbon fluxes
between these two pathways
renders propionibacteria great flexibility to use a broad
spectrum of substrates with various
oxidation states to maintain NADH balance. Since glycolysis
cannot provide enough NADH for
propionic acid production, acetic acid is formed as a
compensating metabolite providing extra
reducing power to maintain redox balance. Consequently, to
produce propionic acid from
glucose, which has a lower redox state (reductance degree = 4)
than propionic acid (reductance
degree = 4.67), requires the co-production of a more oxidized
metabolite acetic acid (reductance
degree = 4). Therefore, propionic acid production from glucose
is tightly coupled with and
limited by acetic acid production. In contrast, glycerol with
the same redox state as propionic
acid (reductance degree = 4.67) has more reducing power than
glucose and its conversion to
pyruvate yields sufficient NADH for propionic acid biosynthesis
without requiring the co-
production of acetic acid to provide additional NADH. When
glycerol and glucose were used as
co-substrates in propionic acid fermentation, they were consumed
simultaneously with glycerol
mainly used for propionic acid biosynthesis and glucose as a
hydrogen donor substrate for the
supply of reducing equivalents and ATP for cell biomass
synthesis (Liu et al., 2011).
Pyruvate is an important node in propionibacteria metabolic
pathways because propionic acid,
acetic acid and succinic acid, as well as biomass, are all
formed from pyruvate. Metabolic flux
analysis was performed to elucidate the carbon flux
distributions at this node for different
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fermentation conditions, and the results are shown in Figure 3.
The flux distribution was
expressed as the percentage of pyruvate formed or consumed in
each pathway. For glucose
fermentation, more than 90% of pyruvate was obtained through the
EMP pathway and less than
10% was through the HMP pathway. In the co-fermentation,
glycerol contributed to ~65% of
pyruvate while glucose only accounted for ~35% (30% via EMP and
5% via HMP), regardless of
the different glycerol/glucose mass ratios (between 1 and 3) in
the fermentation (Fig. 3A). For
the fluxes from pyruvate to various products, about ~75% went to
propionic acid, ~12% to
biomass, ~6.5% to succinic acid, and ~6.5% to acetic acid for
all co-fermentations (Fig. 3B). The
flux to propionic acid was slightly higher at ~80% with glycerol
and much lower at ~52% with
glucose as sole carbon source, whereas the flux to acetate
showed an opposite trend, 3% with
glycerol and 25.6% with glucose as sole carbon source. In
general, the flux toward cell biomass
decreased slightly as more glycerol and less glucose were
present in the fermentation, which was
consistent with the final cell density obtained in the
fermentation. The flux toward succinic acid
did not seem to be affected by the carbon substrate used in
these fermentations. These results
showed that the flux redistribution for redox balance was more
robust in the co-fermentation
with sufficient acetate and ATP biosynthesis to support good
cell growth and faster fermentation
as compared to glycerol fermentation.
3.4 Repeated-batch fermentations in the FBB
Repeated batch fermentations with glycerol and glucose as
co-substrates at 2:1 mass ratio were
studied with cells immobilized in a fibrous-bed bioreactor
(FBB). After a high cell density had
been immobilized in the FBB, three consecutive batches were
performed with glycerol and
glucose in the synthetic medium followed with a fourth batch
with crude glycerol, CB
hydrolysate, and CSL as the substrates. The fermentation
kinetics is shown in Figure 4. In
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general, similar fermentation kinetics was obtained for all four
batches (Fig. 4A), suggesting that
the FBB was stable for continued production of propionic acid
under the repeated batch mode. In
fact, there was a slight increase in both the propionic acid
yield (from 0.52 g/g to 0.58 g/g) and
volumetric productivity (from 0.44 g/Lh to 0.58 g/Lh) from the
first batch to the third batch
(Fig. 4B), a result of increased cell density in the FBB due to
continued cell growth and cell
adaptation (Liang et al., 2012; Suwannakham et al., 2005; Zhang
et al., 2009ab). It is noted that
the OD, an indicative of the density of free cells in the
fermentation broth, in each batch was
significantly lower than that in free-cell fermentation (up to
~10 vs. >15) but the propionic acid
productivity was more than two-fold of that in comparable
free-cell fermentations because of the
higher density of cells immobilized in the FBB. Based on the FBB
working volume, the reactor
productivity was as high as 2.7 g/Lh, which was much higher than
that in free-cell fermentation
at a comparable propionic acid concentration (Fig. 4C).
Comparable propionic acid yield and
productivity were obtained with crude glycerol, CB hydrolysate,
and CSL as the substrates in the
fourth batch, confirming that these low-cost feedstocks can be
used efficiently for propionic acid
production.
3.5 Comparison to other studies
Several studies on propionic acid fermentation with glycerol as
sole carbon source or with a co-
substrate have been reported and are summarized in Table 4 for
comparison. The highest
propionic acid yield of 0.72 g/g from glycerol as sole carbon
source was reported with P.
acidipropionici ATCC 4875 but the productivity was low, only
0.07 g/Lh (Zhang, 2009). Himmi
et al. (2000) reported a good propionic acid yield of 0.64 g/g
and productivity of 0.42 g/Lh from
glycerol with P. acidipropionici ATCC 25562. Ruhal and Choudhury
(2012) reported the
production of propionic acid and trehalose from crude glycerol
using P. freudenreichii subsp.
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15
shermanii, achieving a propionic acid yield of 0.42 g/g with a
significant amount of lactic acid
also produced at a yield of 0.3 g/g. Apparently, the
fermentation performance would be species
and strain dependent. For the same strain, co-fermentation of
glycerol with glucose usually gave
a higher productivity although the propionic acid yield would be
slightly reduced, as shown in
the present study. Recently, Liu et al. (2011) reported
propionic acid yield and productivity of
0.57 g/g and 0.15 g/Lh, respectively, from glycerol/glucose at a
mass ratio of ~2 with P.
acidipropionici ATCC 4965. In our study with the same species
and similar glycerol/glucose
mixture as co-substrates, we obtained comparable propionic acid
yield but a 50% higher
productivity of 0.23 g/Lh. A much higher propionic acid
productivity of 0.58 g/Lh based on
total liquid volume (>2.5 g/Lh based on reactor working
volume) was achieved in the co-
fermentation with the FBB, demonstrating the advantages of the
immobilized-cell fermentation
for long-term continuous production of propionic acid in a
repeated batch mode.
It is noted that the propionic acid yield, productivity, and P/A
and P/S ratios could be
significantly affected by the medium pH. The pKa values of
succinic acid, propionic acid and
acetic acid are 5.6, 4.87 and 4.76, respectively. Most of these
acids are present in the form of
dissociated acids or ions at pH 6.5 while a substantial fraction
of them would be present in the
undissociated form at or near their pKa values. In general, a
lower pH would increase the P/A
ratio because of the reduced cell growth and acetate
biosynthesis. On the other hand, the P/S
ratio was lower in serum bottles without pH control (pH dropped
from 6.8 to 4.8) because of
stronger propionic acid inhibition at the lower pH resulting in
more succinic acid accumulation.
Nevertheless, the glycerol/glucose co-fermentation effects on
cell growth and propionic acid
production were consistent in both the bioreactor with pH
controlled at 6.5 and serum bottles
without pH control (pH 6.8 to 4.8). It should be noted, however,
that the observed co-
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16
fermentation benefits might be species or even strain dependent
as glucose as a co-substrate did
not improve propionic acid production from glycerol by P.
acidipropionici ATCC 4875 (Zhang,
2009).
4. Conclusions
Glucose fermentation produced considerable cell biomass and
acetate, leading to a relatively low
propionate yield, whereas glycerol fermentation had higher
propionate yield and selectivity, but
suffered from low productivity. When glycerol and glucose were
co-fermented, propionate
productivity was greatly improved with higher yield and
selectivity comparable to those of the
glycerol fermentation. Metabolic flux analysis confirmed that
the flux redistribution for redox
balance was more robust in the co-fermentation with sufficient
acetate and ATP biosynthesis to
support cell growth and faster fermentation. Finally, propionate
production from crude glycerol,
cassava bagasse, and corn steep liquor as low-cost feedstocks
was demonstrated.
Acknowledgements
This study was supported in part by a research grant from The
Dow Chemical Company.
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21
Table 1. Stoichiometric equations used in metabolic flux
analysis
Reaction Stoichiometric equation
Glucose oxidation
EMP pathway
HMP pathway
Glucose + 2 ADP + 2 NAD+ 2 Pyruvate + 2 ATP +2 NADH (Eq. 1)
3 Glucose + 5 ADP +11 NAD+ 5 Pyruvate + 3 CO2 + 5 ATP + 11 NADH
(Eq. 2)
Glycerol oxidation Glycerol + ADP + 2 NAD+ Pyruvate + ATP + 2
NADH (Eq. 3)
Organic acids formation from pyruvate
Pyruvate + CO2 + 2 NADH Succinate + 2 NAD+ (Eq. 4)
Pyruvate + ADP + NAD+ Acetate + ATP + NADH + CO2 (Eq. 5)
Pyruvate + 2 NADH + ADP Propionate + 2 NAD+ + ATP (Eq. 6)
Biomass formation 4 Pyruvate + 5.75NADH + 33.7 ATP Biomass +
5.75 NAD+ + 33.7 ADP (Eq. 7)
Equations originally proposed by Papoutsakis and Meyer
(1985)
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22
Table 2. Kinetics of propionic acid fermentation by P. shermanii
in serum bottles.
Substrate Yield (g/g) Productivity
(g/Lh)
P/A ratio
(g/g)
P/S ratio
(g/g)
Gly/Glu
ratio (g/g)
Glucose 0.430.001 0.110.000 2.660.01 9.180.08 -
Glycerol 0.640.013 0.060.003 9.570.38 3.070.10 -
1 Glycerol/Glucose 0.520.011 0.130.001 5.450.32 9.420.16
1.030.02
2 Glycerol/Glucose 0.580.001 0.130.004 5.800.00 7.610.15
1.420.02
3 Glycerol/Glucose 0.610.006 0.120.011 6.690.26 6.760.14
1.520.03
4 Glycerol/Glucose 0.650.005 0.100.003 6.020.01 9.970.02
1.780.08
5 Glycerol/Glucose 0.640.013 0.090.003 6.200.02 7.350.01
1.880.00
Gly/Glu: glycerol consumption rate/glucose consumption rate; The
medium was buffered with 20 g/L CaCO3. During the fermentation, the
pH dropped from the initial value of ~6.5 to the final value of
~4.8. The initial total substrate concentration was 30 g/L. Each
condition was run in duplicated bottles and the average and
standard error are reported.
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23
Table 3. Kinetics of propionic acid fermentation by P. shermanii
in 5-L bioreactor at pH 6.5.
Carbon source Propionate yield
(g/g)
Productivity
(g/Lh)
P/A ratio
(g/g)
P/S ratio
(g/g)
Sp. growth rate
(h-1)
Glucose 0.3900.003 0.1860.007 2.550.05 4.820.15 0.0960.002
Glycerol 0.6470.006 0.1100.001 30.690.08 11.200.21
0.1090.002
1 Glycerol/Glucose 0.5370.006 0.1930.006 13.460.01 9.020.20
0.1020.003
1.5 Glycerol/Glucose 0.5660.003 0.2060.001 15.520.22 9.000.01
0.1080.004
2 Glycerol/Glucose 0.5360.016 0.2280.001 13.510.13 8.170.09
0.1160.005
3 Glycerol/Glucose 0.6450.008 0.1790.001 15.070.16 11.360.19
0.0970.007
2 Glycerol/Glucose 0.5660.001 0.2460.001 8.680.01 5.460.01
0.0980.004
Glucose and glycerol were used as carbon sources in a synthetic
medium with an initial total substrate concentration of 30 g/L. The
last fermentation was with crude glycerol, CB hydrolysate, and CSL
as substrates. For each fermentation, duplicated samples were
analyzed and the average and standard error are reported.
-
24
Table 4. Comparison of propionic acid production from glycerol
as sole carbon source and
glycerol/glucose as co-substrates.
Strain Substrate Propionate yield (g/g) Productivity
(g/Lh) P/A ratio
(g/g) (h-1) Reference
Co-fermentation P. acidipropionici ATCC4965 2 Gly/Glu 0.57 0.15
25.2 - Liu et al., 2011
P. acidipropionici ATCC4875(ack)
3 Gly/Glu 0.41 0.10 13.7 0.13 Zhang, 2009
2 Gly/Glu 0.54 0.23 13.51 0.12
3 Gly/Glu 0.65 0.18 15.07 0.10
P. shermanii DSM4902
Crude glycerol + CB hydrolysate +
CSL 0.57 0.25 8.68 0.10
This study
Glycerol P. acidipropionici ATCC4875
Glycerol 0.77 0.07 - 0.06 Zhang, 2009
0.55 0.026 >100 0.05 P. acidipropionici ATCC4875 (ack)
0.54 0.1 29 0.16
Zhang et al., 2009b
P. acidipropionici ATCC 4965
0.72 0.05 100 0.03 Coral et al., 2008
P. acidipropionici ATCC 25562
~0.68 0.18 45.26 0.08 Barbirato et al., 1997
P. acidipropionici ATCC 25562
~0.64 0.42 ~5.73 0.10*
P. freudenreichii ATCC 9614
~0.47 0.18 ~4.47 0.13*
Himmi et al., 2000
P. shermanii Crude glycerol 0.42 - - - Ruhal and Choudhury,
2012
*: biomass production rate (g/Lh)
-
25
List of Figures
Figure 1. Batch fermentation kinetics of P. shermanii with
glucose (A) or glycerol (B) as sole
carbon source in 5-L bioreactors at pH 6.5, 32 oC.
Figure 2. Batch fermentation kinetics of P. shermanii with
glycerol/glucose mixture as carbon
source at a mass ratio of 2 in synthetic media (A) or with crude
glycerol and cassava
bagasse hydrolysate as carbon source and corn steep liquor as
nitrogen source (B) in a 5-
L bioreactor at pH 6.5, 32 oC.
Figure 3. Metabolic flux distributions in glucose fermentation,
glycerol fermentation, and
glycerol/glucose co-fermentation by P. shermanii.
Figure 4. Kinetics of repeated-batch fermentations in the FBB;
(A) Time course data; (B)
Propionic acid yield and productivity; (C) Effects of propionic
acid titer on volumetric
productivity. Glycerol and glucose at a mass ratio of 2 was used
in the first three batches;
crude glycerol and CB hydrolysate with corn steep liquor were
used in the last batch.
-
26
A
B
Figure 1
-
27
A
B
Figure 2
-
28
A
B
Figure 3
GlucoseEMP
Glycerol
Propionicacid
AceticacidBiomass
Succinicacid
-
29
A
B
C
Figure 4
-
30
Highlights
Glucose gave considerable cell biomass and acetate, with a low
propionate yield
Glycerol gave higher propionate yield and selectivity, but low
productivity
Co-fermentation of glycerol and glucose improved propionate
productivity and yield
Co-fermentation showed robust flux redistribution for redox
balance and cell growth
Propionate can be produced from low-cost crude glycerol and
cassava bagasse