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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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

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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/L·h) 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/L·h). On the

other hand, co-fermentation of glycerol and glucose at an appropriate mass ratio gave both a high

yield (0.54�0.65 g/g) and productivity (0.18�0.23 g/L·h) 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; Martínez-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 32°C 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 50�100 μm 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: 2200–2800 CMC U/g, �-glucosidase activity:

525–775 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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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 24�48 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 24�36 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 45°C 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 7�10, 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/L·h, which was higher than that with glucose (0.19 g/ L·h) and about

double of that with glycerol (0.11 g/L·h) as sole carbon source. The propionic acid yield

(0.54�0.65 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/L·h to 0.58 g/L·h) 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/L·h, 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/L·h (Zhang, 2009). Himmi

et al. (2000) reported a good propionic acid yield of 0.64 g/g and productivity of 0.42 g/L·h 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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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/L·h, 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/L·h. A much higher propionic acid productivity of 0.58 g/L·h based on

total liquid volume (>2.5 g/L·h 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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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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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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Table 2. Kinetics of propionic acid fermentation by P. shermanii in serum bottles.

Substrate Yield (g/g) Productivity

(g/L·h)

P/A ratio

(g/g)

P/S ratio

(g/g)

�Gly/�Glu

ratio (g/g)

Glucose 0.43±0.001 0.11±0.000 2.66±0.01 9.18±0.08 -

Glycerol 0.64±0.013 0.06±0.003 9.57±0.38 3.07±0.10 -

1 Glycerol/Glucose 0.52±0.011 0.13±0.001 5.45±0.32 9.42±0.16 1.03±0.02

2 Glycerol/Glucose 0.58±0.001 0.13±0.004 5.80±0.00 7.61±0.15 1.42±0.02

3 Glycerol/Glucose 0.61±0.006 0.12±0.011 6.69±0.26 6.76±0.14 1.52±0.03

4 Glycerol/Glucose 0.65±0.005 0.10±0.003 6.02±0.01 9.97±0.02 1.78±0.08

5 Glycerol/Glucose 0.64±0.013 0.09±0.003 6.20±0.02 7.35±0.01 1.88±0.00

�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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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/L·h)

P/A ratio

(g/g)

P/S ratio

(g/g)

Sp. growth rate

μ (h-1)

Glucose 0.390±0.003 0.186±0.007 2.55±0.05 4.82±0.15 0.096±0.002

Glycerol 0.647±0.006 0.110±0.001 30.69±0.08 11.20±0.21 0.109±0.002

1 Glycerol/Glucose 0.537±0.006 0.193±0.006 13.46±0.01 9.02±0.20 0.102±0.003

1.5 Glycerol/Glucose 0.566±0.003 0.206±0.001 15.52±0.22 9.00±0.01 0.108±0.004

2 Glycerol/Glucose 0.536±0.016 0.228±0.001 13.51±0.13 8.17±0.09 0.116±0.005

3 Glycerol/Glucose 0.645±0.008 0.179±0.001 15.07±0.16 11.36±0.19 0.097±0.007

2 Glycerol/Glucose 0.566±0.001 0.246±0.001 8.68±0.01 5.46±0.01 0.098±0.004

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.

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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/L·h)

P/A ratio (g/g) μ (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/L·h)

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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.

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A

B

Figure 1

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�A

B

Figure 2

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A �

B

Figure 3

Glucose�EMP

Glycerol

Propionic�acid

Acetic�acidBiomass

Succinic�acid

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A

B�

C�

Figure 4

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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�