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: yang.15@osu.edu




2

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




3

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 B
12
(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



4

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.




5

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 K
2
HPO
4
, 0.05g MnSO
4
, 20 g CaCO
3
, 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.



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 (OD
600
~3.0)
in NLB medium in a serum tube. The serum bottle cultures were incubated at 32
o
C with pH
buffered with 20 g/L CaCO
3
, (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 N
2
for 45 min to



7

anaerobiosis, the fermentor was inoculated with 50 ml of an overnight culture (OD
600
~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



8

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 H
2
SO
4
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



9

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 + CO
2
+ H
2
O
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 + H
2
O
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



10

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



11

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



12

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



13

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



14

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.



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 pK
a
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 pK
a
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-



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 CO
2
+ 5 ATP + 11
NADH (Eq. 2)
Glycerol oxidation Glycerol + ADP + 2 NAD
+
Pyruvate + ATP + 2 NADH (Eq. 3)
Organic acids
formation from
pyruvate
Pyruvate + CO
2
+ 2 NADH Succinate + 2 NAD
+
(Eq. 4)
Pyruvate + ADP + NAD
+
Acetate + ATP + NADH + CO
2
(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)



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 CaCO
3
. 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.



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
o
C.
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
o
C.
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
Glucose EMP
Glycerol
Propionic acid
Acetic acid
Biomass
Succinic acid



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