Biochemical Engineering Journal 192 (2023) 108816

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Biochemical Engineering Journal

journal homepage: www.elsevier.com/locate/bej

Regular article

Propionic acid production via two-step sequential repeated batch

fermentations on whey and flour
Emine Bezirci *, 1, Hatice Taşpınar-Demir 1, Burcu Turanlı-Yıldız 1, Atacan Erdem , Filiz Alemdar ,
Mustafa Türker
Pak Gıda Üretim ve Pazarlama A.Ş., Köseköy Mahallesi Ankara Caddesi No:277 Kartepe, Kocaeli, Turkey

A R T I C L E I N F O A B S T R A C T

Keywords: In this study, two-stage process was developed for propionic acid production via repeated batch and fed-batch
Two-step fermentation fermentations. In the first step, either whey lactose or flour hydrolysate was used as carbon source to produce
Lactobacillus plantarum lactic acid using Lactobacillus plantarum in repeated batch and fed-batch fermentations. Then, produced lactic
Propionibacterium freudenreichii
acid was used as a carbon source for propionic acid fermentation using Propionibacterium freudenreichii in
Whey
Wheat flour
repeated batch fermentations. Maximum lactic acid concentrations were achieved as 100 g/L at 250th hour of
Repeated batch fermentation incubation (corresponding to 6th cycle of repeated batch fermentation) on whey lactose and 97 g/L at 500th
hour (1st cycle) on flour hydrolysate as substrate. For propionic acid production, maximum propionic acid
concentrations were obtained as 28 g/L at 700th hour (13th cycle) on lactic acid obtained from whey and 35 g/L
at 3600th hour (10th cycle) on lactic acid obtained from flour hydrolysate. Lactic acid and propionic acid
productivities were 0.80 g/Lh and 0.050 g/Lh on whey lactose higher than the values 0.12 g/Lh and 0.025 g/Lh
obtained on flour hydrolysate respectively.

1. Introduction environmental concerns, consumer’s bio-based demands [7].

Propionic acid, also known as propanoic acid, is a short-chain fatty
acid in “generally recognized as safe (GRAS)” status. It has various in­
dustrial uses such as mold inhibition and fruit flavouring in food in­
dustry, feed and grain preservation, as perfume bases in cosmetics, in
cellulose based plastic manufacturing in plastics industry, in veterinary
applications for wound infections, in pharmaceutical industry for
conjunctivitis, anti-arthritic drugs and also for herbicide production
[1–4].
In the world market, propionic acid demand was 470,000 tons in
2019, which is expected to reach 550,000 tons in 2026 [5]. Global prices
range between 2 and 3 USD/kg. The industrial production of propionic
acid is mainly performed by petrochemical route via Reppe or Larson
processes [6]. In food industry, propionic acid has been used as food
preservative in the form of calcium propionate obtained by petro­
chemical route. However, in recent years there is a growing interest to
use propionic acid produced by fermentative route on agricultural and
bio-based feedstocks due to the depletion of fossil-based resources,
Several microorganisms such as Propionibacteria, Clostridium, Fuso­
bacterium, Veillonella, Selenomonas genera are capable of making
fermentative propionic acid production. Propionibacteria is the most
widely used genus producing propionic acid via dicarboxylic acid
pathway [8,9]. These microorganisms have a capability to use different
types of cheap industrial and agricultural by-products as a substrate to
grow and produce propionic acid [10]. Propionibacterium freudenreichii is
one of them and it has a good potential for producing propionic acid.
The first study on propionic acid production by Propionibacteria dates
back to 1878 by Albert Fitz, revealing Fitz Equation, where 2 moles of
propionic acid, 1 mol of acetic acid, 1 mol of CO2 and 1 mol H2O are
produced per 3 moles of lactic acid. First complete study of propionic
acid metabolism was performed in 1937 [6]. The studies illuminating
the genetic background of Propionibacteria strains were released more
recently [11–14].
The major limitation of fermentative propionic acid production is its
cost compared to petrochemical route. Due to low growth rates and
productivity levels, some of microbial processes currently are not able to

* Corresponding author.
E-mail addresses: emine.bezirci@pakmaya.com (E. Bezirci), hatice.demir@pakmaya.com (H. Taşpınar-Demir), turanli@gmail.com (B. Turanlı-Yıldız), atacan.
erdem@pakmaya.com (A. Erdem), filiz.alemdar@pakmaya.com (F. Alemdar), mustafa.turker@pakmaya.com (M. Türker).
1
These authors contributed equally to this manuscript.

https://doi.org/10.1016/j.bej.2023.108816
Received 9 November 2022; Received in revised form 25 December 2022; Accepted 16 January 2023
Available online 19 January 2023
1369-703X/© 2023 Elsevier B.V. All rights reserved.
E. Bezirci et al.
compete with the petrochemical routes in terms of production costs.
Moreover, co-production of acetic acid and succinic acid leads to
complicated and costly downstream processing when high degree of
purity is needed. However, there is an upward trend in food industry to
use products produced by fermentative routes as natural preservatives.
This tendency provokes further investigations on fermentation strategies
to achieve higher production rates from low-cost carbon sources [2,9].
Process optimization studies for propionic acid on several fermen­
tation modes such as batch [8,9,15], fed-batch[7,9], continuous [16],
semi-continuous [17], extractive fermentation [18,19] and cell immo­
bilized culture systems [16,20] were investigated. Batch fermentation
process causes some bioprocess limitations because of substrate and
product inhibition, slow growth rates, low product titres, yields and
productivities [21].
Microbial production of propionic acid is performed commonly using
monoculture processes. Recently, the potential of the co-culture process
has been considered for propionic acid production [22]. Co-cultivation
of lactic acid and propionic acid bacteria is a common application. For
this application, lactic acid bacteria produces lactic acid, which acts as a
substrate for propionic acid bacteria to grow and produce propionic acid
[23]. Sabra et al. showed the production of propionic acid by
co-cultivation of lactic acid and propionic acid bacteria through the
conversion of lactate to propionic acid using fed-batch strategy.
Lactic acid, has a potential to be used as a substrate for microbial
production of propionic acid and is used in a range of industrial and
biotechnological applications such as food, pharmacy, cosmetics and
chemistry [24,25]. Commercial lactic acid production is performed
either chemically by the hydrolysis of lactonitrile or by bacterial
fermentation. Annually 90 % of total production is made by bacterial
fermentation [26]. Lactic acid bacteria are robust microorganisms under
stress factors such as high acid concentration and are able to metabolize
various carbon sources such as whey, molasses, starch, lignocellulose,
wheat bran and flour [27]. Whey is the main by-product of dairy in­
dustry that contains approximately 60–65 % (w/v) of lactose, milk nu­
trients, minerals and vitamins [28,29]. It has a potential as a substrate
for industrial processes and is used as substrate for bacterial lactic acid
fermentation [29].
Lactic acid bacteria can metabolize sugars via different pathways
resulting in homo-, hetero- or mixed acid fermentations. In homo­
fermentation, it produces only lactic acid via Embden-Meyerhof-Parnas
pathway; while in heterofermentation, equimolar amounts of lactic acid,
carbon dioxide and ethanol or acetate are produced via the phospho­
ketolase pathway. Mixed acid fermentation is another process, where
six-carbon sugars are converted into complex and different mixture of
acids [26]. Lactobacillus plantarum is a facultative heterofermentative
bacterium that produces lactic acid on hexoses, while producing lactic
acid and acetic acid on pentoses [30]. Moreover, L. plantarum is one of
the most acid tolerant bacteria and it is used for production of lactic acid
from different lactose containing substrates [31].
P. freudenreichii cannot use glucose and/or lactose to make propionic
acid. Therefore, a two-step fermentation strategy was developed for
microbial propionic acid production in repeated batch mode on both
whey and flour hydrolysate as substrates. The first step involved lactic
acid production via L. plantarum using either whey lactose or wheat flour
hydrolysate. At the second step, propionic acid was produced via
P. freudenreichii on lactic acid that was obtained in the previous step.
This two-stage process is designed to use bio-based resources as a sub­
strate to obtain bio-based propionic acid that can be used as a food
ingredient.
2. Materials and methods
2.1. Materials
Unless otherwise specified, all chemicals were analytical grades and
purchased from Sigma-Aldrich (USA) or Merck (Germany).
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Biochemical Engineering Journal 192 (2023) 108816
2.2. Strains and cultivation conditions
L. plantarum (Maysa Gıda San. Tic. A.Ş., Türkiye) and P. freudenreichii
(DSMZ-20271) strains were used throughout the study. Microbank (Pro-
Lab Diagnostics, UK) was used for long-term storage of the cultures at
− 80 ◦ C. MRS agar / broth (Merck, Germany) were used for cultivation of
L. plantarum. For the bioreactor inoculum, cells were inoculated from
agar plates to MRS broth and incubated at 30 ◦ C for 24 h. Propionibacter
Isolation Agar Base (HiMedia, India) was used for cultivation of
P. freudenreichii. The bioreactor inoculum was prepared with the incu­
bation of agar grown cells cultivation broth at 30 ◦ C for 72 h.
2.3. Whey preparation
Commercial whey powder (Milkon, Türkiye) containing 85–90 %
lactose was used for the study. Feeding whey solution was prepared at a
concentration of 200 g/L.
2.4. Flour hydrolysis
Regular wheat flour with a dry matter of 85.5 %, a protein content of
10.5 %, and an ash content of 0.7–0.8 % was used in this study. Wheat
flour solution (25 %, w/v) was digested using 160 ppm α-amylase from
Bacillus amyloliquefaciens (BAN) for 3 h at 70 ◦ C at pH 6.0 and with 320
ppm glucoamylase (AG XXL) for 24 h at 60 ◦ C at pH 4.5. The hydrolysate
was pasteurized for 30 min at 85 ◦ C.
2.5. Fermentation process
A two-step repeated batch fermentation strategy was adopted
(Fig. 1). Lactic acid fermentation by L. plantarum was followed by pro­
pionic acid fermentation by P. freudenreichii. The final product of the
first fermentation process was used as the carbon source in the second
step. The carbon source in the lactic acid fermentation was either whey
or flour hydrolysate.
2.5.1. Lactic acid production
Lactic acid fermentation protocols are explained in two categories
based on the carbon source used (either whey or flour hydrolysate).
2.5.1.1. Whey as the substrate. Lactic acid fermentations on whey was
carried out at 35 ◦ C in 16 L stainless steel stirred tank bioreactor
(NFL22, Bioengineering, Switzerland) with an initial volume of 6 L.
Bioreactor inoculation ratio was used as 10 % (v/v) of the total volume.
The cells used for inoculation were produced in MRS broth. Lactic acid
production medium was consisted; 45 g/L whey powder, 5 g/L yeast
extract, 0.05 g/L MnSO4.4 H2O, 0.5 g/L MgSO4.7 H2O, 2.5 g/L
(NH4)2HPO4 [32], with a feed whey concentration of 65 g/L and
150–200 g/L for optimization and repeated batch studies, respectively.
Optimization studies were conducted in batch, fed-batch and pulse feed
operation modes, where a total of 260 g whey was fed for the fed-batch
and pulse feed operations. For fed-batch operation, linear feeding was
applied between 10.5 and 22th hour, while one shot feeding was applied
at 12th hour for pulse feeding. For each repeated cycle, 2 L of the
fermentation broth, approximately 16 % of the final volume, was left in
the bioreactor, and new medium was added in a manner to obtain same
initial conditions under sterile conditions. Whey solution was fed to the
system when initial lactose was depleted. pH was adjusted to pH 5.5 by
addition of Na2CO3 (15 %, w/v), stirrer speed was set to 300 rpm and no
aeration was used.
2.5.1.2. Flour hydrolysate as the substrate. Lactic acid fermentation on
flour hydrolysate was carried out in BIOSTAT B benchtop system
(Sartorius, Germany) at 30 ◦ C, 200 rpm in a 3 L vessel with 1.5 L
working volume. pH was adjusted to 5.5 with Na2CO3 (15 %, w/v).
E. Bezirci et al.
Fig. 1. Two-step repeated batch fermentation strategy for propionic acid production on lactic acid.
When glucose was depleted in the media, half of the fermentation broth
was taken to feed the propionic acid bioreactor, thereafter a new batch
was started with the addition of sterile flour hydrolysate.
P. freudenreichii is not able to use glucose to make propionic acid (data
not shown). Therefore, flour hydrolysate was first converted to lactic
acid to be used as a feedstock in the second step to make propionic acid.
2.5.2. Propionic acid production
Propionic acid fermentation protocols are also categorized based on
the initial carbon source used in the lactic acid fermentation step
previously:
2.5.2.1. Lactic acid obtained from whey as the substrate. For propionic
acid fermentations on whey, 100 L stainless steel stirred tank bioreactor
(Bioengineering, Switzerland) was used with a working volume of
35–85 L. Inoculum for 100 L bioreactor was prepared via a two-step
process: first shake-flask cultivations were transferred to 16 L biore­
actor, thereafter cells were transferred to 100 L bioreactor with an
inoculation ratio of 15 % (v/v) of the total volume. Propionic acid fer­
mentations were conducted in a medium containing; 12 L lactic acid
(33.75 g/L) which was produced on whey, 8.6 g/L casein enzymic hy­
drolysate, 8.6 g/L yeast extract, 0.087 g/L MgSO4.7 H2O and 0.22 g/L
K2HPO4. Reactor was operated in repeated batch mode where lactic acid
was fed to the system. Prior to feeding, certain volume of propionic acid
fermentation broth was discharged and then equal volume of the lactic
acid feed from first stage was fed to propionic acid bioreactor. The pH
was adjusted to 6.0 using NaOH (18 %, w/v) and temperature was set to
30 ◦ C, where stirrer speed was set to 50 rpm with no aeration. N2 flow
was supplied for at least 15 min at the beginning of the fermentation in
order to maintain anaerobic conditions. Afterwards, dissolved O2 levels
were monitored and fermenter was sparged with N2 to maintain dis­
solved O2 levels below 5 %.
2.5.2.2. Lactic acid obtained from flour hydrolysate as the substrate.
Propionic acid fermentation on flour hydrolysate was conducted using
BIOSTAT B benchtop system (Sartorius, Germany) in a 3 L vessel with
1.5 L working volume. The settings were as follows; temperature 32 ◦ C,
initial pH 6.5 and adjusted to pH 6.0 after 87 h, agitator ran at 50 rpm,
no air inlet. N2 gas was supplied for at least 15 min at the beginning of
the fermentation. Dissolved O2 levels were monitored throughout the
fermentation and fermenter was sparged with N2 to maintain dissolved
O2 levels below 5 %. Fermentations were started with a synthetic
lactate-based media, which contained; 10 g/L Na-lactate, 10 g/L casein
enzymic hydrolysate, 10 g/L yeast extract, 0.1 g/L MgSO4.7 H2O,
3
Biochemical Engineering Journal 192 (2023) 108816
0.25 g/L K2PO4. Reactor was operated in repeated batch mode where
lactic acid was fed to the system. The bioreactor was run with synthetic
lactate for the first 973 h; afterwards lactic acid from flour hydrolysate
was used. According to the lactic acid concentration in the media, lactic
acid was fed to the bioreactor, where the same amount fermentation
broth was taken out and cells were recycled in sterile conditions.
2.6. Analytical methods
Organic acids were determined with HPLC using Hi-Flex H,
300 mm × 7.7 mm column (Agilent Technologies) with 0.6 mL/min
flow rate at 55 ◦ C column temperature and 5 mM H2SO4 as mobile
phase. The organic acids (lactic acid, propionic acid and acetic acid)
were detected by diode array detector. Glucose and lactose were
determined using with HPLC with a Sugar SH1011, 300 mm × 8.0 mm
column (Shodex) with 0.8 mL/min flow rate at 50 ◦ C column tempera­
ture using 5 mM H2SO4 as mobile phase with refractive index detector.
Osmotic pressure was determined with Osmomat 030 Automatic Cryo­
scopic Osmometer (Gonotec, United States). Dry matter determination
was performed using MA150 Moisture Analyzer (Sartorius, Germany).
Optical density of the cells was measured at 600 nm using UV/Vis
Spectrophotometer (Shimadzu, Japan). The number of colony forming
units (CFU) was determined after 2 days of incubation at 30 ◦ C on MRS
(de MAN, ROGOSA and SHARPE) agar for L. plantarum, on HiMedia
Propionibacter isolation agar base for P. freudenreichii. The number of
viable yeast colonies were count on YM (yeast and mold) agar plates
after incubation 48 h at 30 ◦ C and expressed as colony forming units per
mL (CFU/mL).
3. Results and discussion
3.1. Lactic acid fermentations
Batch, pulse feed and fed-batch fermentation protocols were inves­
tigated to improve lactic acid production using whey lactose as the
substrate in a 16 L bioreactor. Feedings were conducted when lactose
was depleted in the bioreactor. For pulse feeding, 4 L of whey solution
was fed to the system at the 12th hour, while for fed-batch case linear
feeding was applied between 10.5 and 22th hour according to the
feeding profile given in Fig. 2. For both strategy, a total of 260 g whey
was fed to the bioreactor (Fig. 2). For these three modes of fermentation,
maximum lactic acid concentrations, maximum cell dry weights,
maximum lactic acid productivities, biomass (YX/S) and product (YP/S)
yields were listed in Table 1. When compared with batch fermentation
E. Bezirci et al. Biochemical Engineering Journal 192 (2023) 108816

fermentation. Additionally, 1.1 g/L acetate was produced along with

lactic acid. The productivity levels were initially at 1.61 g/Lh at rela­
tively low lactic acid concentrations then decreased to 1.05 g/Lh when
lactic acid concentrations reached to 100 g/L, averaging 1.23 g/Lh
(Table 2). There is no enough data between 200 and 260th hours (6th
cycle) to calculate the productivity. Probably lactic acid was finished
earlier. Therefore, we have skipped productivity data for this cycle in the
table. However, product yields (YP/S) remained constant very close to
thermodynamic maximum (1.05 g lactic acid/g lactose) at each cycle.
The maximum thermodynamic yield is defined as the ratio of degree of
reduction of lactose to the degree of reduction of lactic acid. However,
the productivities were decreased as the final lactic acid concentrations
increased in each cycle, possibly due to the product inhibition. Then,
lactic acid produced at the end of each cycle was used as substrate in the
following propionic acid fermentations.
After the trials with whey, repeated batch fermentations were con­
ducted using flour hydrolysate with ~130 g/L of initial glucose con­
centration. Fig. 4 shows glucose consumption and lactic acid production
levels during a total of 1881 h of fermentation. Each cycle was continued
until glucose was depleted in the media and resulted in 75 – 95 g/L lactic
acid production. The product yields and productivity values for each
cycle were given in Table 3. The glucose consumption rates were higher
at the initial stage of the conversion process and then slowed down due
to lactic acid accumulation. Lactic acid productivity was around 0.24
± 0.07 g/Lh until 60 g/L lactic acid concentrations, beyond this con­
centration it decreased to 0.08 ± 0.01 g/Lh as shown in Table 3. The
product yields at initial cycles were close to maximum thermodynamic
yield (1 g lactic acid/g glucose) however; it gradually dropped
throughout the process, which could be due to possible yeast contami­
nation coming from flour despite heat treatment of the hydrolysate.
Microbial composition of the culture was monitored in propionic acid
fermentation as shown in Fig. 9.
Available evidence supports the view that stress conditions might
lead to biphasic growth and fermentation profiles in Lactobacillus strains
[33] and 60 g/L lactic acid is the growth limiting concentration in batch
Fig. 2. Biomass ( ), lactose ( ), lactic acid ( ), volume ( ) and feed ( ) cultures [34]. Our results shown in Table 3 are coherent with this view.
results of batch (A), fed-batch (B) and pulse feed (C) modes of operation for Lactic acid fermentations were initiated using 4 × 107 CFU/mL lactic
lactic acid production using whey.
acid bacteria. For cell reuse, 1:2 – 1:3 of the fermentation broth was
transferred to the next cycle. Number of viable cells per millilitre were
Table 1
relatively steady and ranged 106 to 108 throughout the process.
Biomass and lactic acid production, yield and productivity values of batch, fed- Fermentation broths collected at the end of each cycle were sterilized
batch and pulse feed modes of operation for lactic acid production using whey and that were used as substrate as lactic acid source for subsequent
lactose. propionic acid fermentations.
Fermentation Lactic Cell dry Biomass Product Productivity
mode acid weight yield, yield, (g/Lh) 3.2. Propionic acid fermentations
(g/L) (g/L) YX/S YP/S
(g/g) (g/g) Propionic acid fermentation on lactic acid obtained from whey was
Batch 29.15 4.22 0.13 0.86 1.43 performed in a 100 L bioreactor with a working volume ranging from
Fed-batch 40.57 4.76 0.09 0.92 1.98 35 L to 85 L by repeated batch fermentation strategy. This repeated
Pulse feed 34.47 4.77 0.12 0.86 2.23
batch fermentations continued about 2000 h and propionic acid, acetic
YX/S; the ratio of total biomass produced (g) to total lactose consumed (g), YP/S; acid and lactic acid concentrations were monitored as shown in Fig. 5.
the ratio of total lactic acid produced (g) to total lactose consumed (g), pro­ Fermentation resulted with a maximum production of 28.70 g/L pro­
ductivity; maximum lactic acid productivity on lactose. pionic acid at 688 h. Acetic acid was detected throughout the fermen­
tation but there was no remarkable change. The maximum number of
results, feeding strategies seem to improve final lactic acid titer and viable propionic acid bacteria achieved was 1.43 × 109 CFU/mL at
product yields, channelling more lactose into lactic acid rather than 188th hours, there after slight decrease was achieved throughout the
biomass. These could be possibly due to more lactose converted to lactic fermentation most probably due to the product inhibition (data not
acid at high lactic acid concentrations due to the acid stress when fed- shown in graphs). Additionally, osmotic pressure values were also
batch phase started. reached to 2 osmol/kg which has a negative effect on growth (Fig. 6).
Fig. 3 shows the lactic acid and lactose concentrations of repeated The productivity levels were in the range of 0.05 ± 0.03 g/Lh.
batch fermentation on whey. The number of viable lactic acid bacteria Repeated batch fermentation on lactic acid obtained from flour hy­
was approximately 5.5 × 109 CFU/mL throughout the fermentation. drolysate was adopted for propionic acid production process using
During the repeated processes, lactic acid concentrations increased P. freudenreichii. Lactic acid levels, propionic acid and acetic acid pro­
to around 100 g/L despite the challenging conditions due to high lactic ductions are shown in Fig. 7.
acid concentrations and high osmotic pressure values. The maximum Propionic acid fermentation was started at pH 6.5 using initial 10 g/
concentration of lactic acid was obtained as 102 g/L after 270 h of L synthetic lactic acid as substrate, after 87th hours the pH set to 6.0

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E. Bezirci et al. Biochemical Engineering Journal 192 (2023) 108816

Fig. 3. Lactic acid production ( ) and lactose consumption ( ) levels during repeated batch fermentation on whey.

concentration reached to about 35 g/L. However, lactic acid consump­

Table 2
tion rates gradually decreased over time as shown by the change in the
Lactic acid concentration, product yield (YP/S) and productivity values for lactic
slopes of lactic acid consumption curves (Fig. 7). The yields and pro­
acid fermentation on whey.
ductivities for propionic acid were calculated based on feeding solutions
Cycle Lactic acid Product yield (YP/ Productivity (g/
shown in Table 4.
number concentration S) Lh)
(g/L) (g lactic acid/g
The osmotic pressure values were presented in Fig. 8. When Fig. 8
lactose) and Fig. 7 were compared, it is remarkable that osmotic pressure levels
correlate with the propionic acid concentrations in the medium. Gradual
Cycle 1 46.60 1.12 1.61
Cycle 2 74.97 1.10 1.39 built up of osmolarity (propionic acid concentration) was observed and
Cycle 3 77.95 1.20 1.26 resulted in increased stress on the microorganisms as shown by the slope
Cycle 4 79.24 0.97 0.96 of propionic acid production curves. Therefore, to improve the pro­
Cycle 5 90.42 1.11 1.25
ductivities and to reduce the fermentation time osmolarity should be
Cycle 7 98.90 1.18 1.05
Cycle 8 98.36 1.09 1.12
monitored and reduced.
average - 1.11 1.23 Comparison of productivity values both for lactic acid and propionic
acid fermentations on whey and flour hydrolysate are shown in Table 5.
YP/S; the ratio of total lactic acid produced (g) to total lactose consumed (g),
The productivity levels on whey are significantly higher than those on
productivity; maximum lactic acid productivity on lactose.

where a steep rise in propionic acid production was observed. After the Table 3
lactic acid in the medium was consumed, concentrated synthetic lactic Yield (YP/S) and productivity values of lactic acid production on flour
acid was added and brought to the initial concentration. This process hydrolysate.
was repeated twice in total until the 973th hour and no change in vol­ Yield, YP/S Productivity levels (g/Lh)
ume occurred. In this process, biomass increase was achieved and lactic (g lactic acid/g
< 60 g lactic > 60 g lactic Totala
acid inhibition was prevented. At the 973rd hour, 300 mL of medium glucose)
acid/L acid/L
was removed and the cells were separated under sterile conditions and
Cycle 1 1.01 0.23 0.08 0.14
transferred back into the fermenter and then, 300 mL lactic acid solu­
Cycle 2 0.99 0.31 0.09 0.16
tion, produced from flour hydrolysate, was added to the medium. These Cycle 3 0.62 0.26 0.06 0.09
additions were repeated after lactic acid concentrations in the broth Cycle 4 0.57 0.15 0.07 0.10
dropped below 1 g/L. These repeated cycles continued until about Averageb 0.80 0.24 ± 0.07 0.08 ± 0.01 0.12
5000 h. It has been observed that around 10 g/L lactic acid concentra­ ± 0.03

tions are optimal for propionic acid production. Therefore, the a

Indicates the productivity of cycle 1, 2, 3, 4 at the point where the lactic acid
maximum lactic acid concentrations in the medium were kept at less concentration is greater than or less than 60 g/L; baverage productivity per
than 20 g/L in repeated cycles. The maximum propionic acid cycle. Presented as the mean ± standard deviation.

Fig. 4. Lactic acid production ( ) and glucose consumption ( ) levels during repeated batch fermentation on flour hydrolysate by L. plantarum.

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E. Bezirci et al. Biochemical Engineering Journal 192 (2023) 108816

Fig. 5. Lactic acid ( ), propionic acid ( ) and acetic acid ( ) levels during the propionic acid fermentation with lactic acid obtained on whey.

Fig. 6. Osmotic pressure values during the propionic acid fermentation with lactic acid obtained on whey.

Fig. 7. Lactic acid ( ), propionic acid ( ) and acetic acid ( ) levels during repeated batch fermentation process with synthetic lactic acid and lactic acid obtained on
flour hydrolysate.

flour. Being semi-solid feedstock, flour is a difficult raw material to be
used as a substrate in microbial fermentations due to its solubility and
heterogeneity. It is preferred as it is a natural substrate but the
contamination risk is high due to sterilization challenges. During pro­
pionic acid fermentation on flour hydrolysate, yeast, lactic acid and
propionic acid bacteria populations and alcohol concentrations were
monitored as an indicator and the results are shown in Fig. 9. The
alcohol concentrations were observed early in the fermentations prob­
ably due to the slow hydrolysis of unhydrolyzed starch and yeast cells
not killed during the sterilization process. However, propionic acid
production still continued despite the presence of yeast cells because
P. freudenreichii cannot use glucose as a substrate and did not compete
with yeast cells for glucose.
The maximum thermodynamic yield of propionic acid is 0.70 g
propionic acid/g lactic acid. When lactic acid is used as the carbon
source, P. freudenreichii produces by-product volatile fatty acids such as
acetic acid and the yield decreases below 0.70 g propionic acid/g lactic
acid. On the other hand, when flour hydrolysate was used, the product
yields in the first and second stage were lower probably due to presence
of yeast in the flour despite sterilization. This resulted in alcohol for­
mation in the second stage as well as justified by the microbial counts as
shown in Fig. 9. Table 6 summarizes the kinetic and stoichiometric
parameters of propionic acid fermentation in this study.
Table 7 summarizes propionic acid production on different carbon

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E. Bezirci et al. Biochemical Engineering Journal 192 (2023) 108816

Fig. 8. Osmotic pressure values during the propionic acid fermentation with synthetic lactic acid (0–973 h) and lactic acid obtained on flour hydroly­
sate (973–4000 h).

Fig. 9. The number of propionic acid bacteria ( ), lactic acid bacteria ( ), yeast colonies ( ) and ethanol ( ) concentration during the propionic acid fermentation
on flour hydrolysate.

Table 4 Table 6
Yield (YP/S) and productivity values for propionic acid in repeated batch Productivity and yield (YP/S) values for propionic acid fermentation on different
fermentations. carbon sources by P. freudenreichii.
Time period Yielda, YP/S Productivityb Substrate Propionic acid productivity Propionic acid yield, YP/S
(h) (g propionic acid/g (g/Lh) (g/Lh) (g/g)
lactic acid)
lactic acid 0.050 ± 0.03 0.53 ± 0.09
Synthetic lactic 0–973 0.507 ± 0.11 0.016 (from whey)
acid ± 0.005 lactic acid 0.025 ± 0.03 0.44 ± 0.14
Flour based lactic 981–4549 0.440 ± 0.13 0.012 (from flour
acid ± 0.009 hydrolysate)
a
The ratio of total propionic acid produced (g) to total lactic acid consumed
(g), sources using Propionibacterium. Immobilization or fed-batch fermenta­
b
Propionic acid productivity on lactic acid. Presented as the mean ± standard
tion processes were preferred in order to untangle substrate inhibition
deviation.
limitations. Additionally, adopting repeated batch fermentation is an
advantageous method for elimination of slow growth and substrate in­
Table 5 hibition, which also enables cell reuse.
Productivity values for propionic and lactic acid fermentations on whey and Acid tolerance of the strains used for propionic acid fermentation is a
flour hydrolysate. crucial aspect. As shown in Table 7, Propionibacterium, having the
capability to produce propionic acid using several cheap wastes (such as
Lactic acid productivitya (g/ Propionic acid productivityb (g/
Lh) Lh) cornstalk, whey, etc.) as a substrate [4,16,35,38], is valuable in terms of
the economical point of view. When suitability for food industry is also
Whey 0.80 ± 0.25 0.050 ± 0.03
Flour 0.12 ± 0.03 0.025 ± 0.03 taken into consideration, whey lactose and flour hydrolysate becomes
hydrolysate prominent and advantageous.
a There is a wide range on productivity and yield values for propionic
Lactic acid productivity on lactose (whey) / glucose (flour hydrolysate),
b acid production, depending on the type of the process and substrates
propionic acid productivity on lactic acid. Presented as the mean ± standard
deviation. (Table 7). There is a limited knowledge on using whey lactose and flour
hydrolysate for propionic acid production in the literature. According to
literature reports and the data obtained in this study, the modes of
operation has an important role to overcome product inhibition. Li et al.

7
E. Bezirci et al. Biochemical Engineering Journal 192 (2023) 108816

Table 7
Productivity and yield (YP/S) values and maximum concentration for propionic acid fermentation on different carbon sources by Propionibacterium.
Microorganism (s) Substrate Fermentation type Propionic acid Propionic acid Maximum propionic acid Reference
productivity (g/Lh) yield, YP/S concentration
(g/g) (g/L)

Propionibacterium flour hydrolysate batch - 0.63 37.7 [2]

acidipropionici
Propionibacterium soy molasses sequential batch 0.42 0.46 21.2 [4]
acidipropionici fermentations
Lactobacillus zeae & Veillonella flour hydrolysate fed-batch 0.33 - 30.0 [9]
criteci (coculture)
Propionibacterium flour hydrolysate batch ~0.14 - ~11.5
acidipropionici
Propionibacterium glycerol high-cell-density - - 50.8 [10]
acidipropionici sequential batches
Propionibacterium whey recycle batch 0.23 0.40 65.0 [16]
acidipropionici (immobilize cell
(immobilized) bioreactor)
Propionibacterium freudenreichii cornstalk hydrolysates expanded bed 0.35 0.75 91.4 [35]
adsorption bioreactor
Propionibacterium apple pomace batch 0.06 0.38 7.7 [36]
freudenreichii (flask)
Propionibacterium lactic acid batch 0.07 0.62 - [37]
acidipropionici (from food waste & waste (fibrous bed bioreactor)
activated sludge)
Propionibacterium whey batch 0.20 0.45 27.0 [38]
acidipropionici fed-batch 0.50 0.62 125.0
(fibrous bed bioreactor)
Propionibacterium freudenreichii glucose fed-batch 0.16 - 34.0 [39]
Propionibacterium glucose batch 0.12 0.36 14.6 [40]
freudenreichii fed-batch (multi-point 0.14 0.43 67.0
fibrous-bed
bioreactor)

aimed production of propionic acid from food waste including lactate.
To prevent lactate inhibition, repeated batch fermentation using fibrous
bed reactor was performed. Substrate inhibition is a critical point for
these fermentations. Therefore, we used also repeated batch process in
our study. Jiang et al. obtained maximum 125 g/L propionic acid after
120 h with using fibrous bed bioreactor by fed-batch fermentation in
spite of 27 g/L propionic acid was obtained with using free cells by batch
fermentation. Dishisha et al., performed high-cell-density sequential
batches with cell recycle using P. acidipropionici on glycerol. Propionic
acid concentration reached at 50.80 g/L. The results showed that, fed-
batch fermentation combined with cell retention systems may improve
propionic acid productivities and titers.
Sabra et al. have developed a process to produce propionic acid using
P. acidipropionici and V. criteci co-culture on flour hydrolysate. Maximum
30 g/L propionic acid was obtained with 0.33 g/Lh productivity by fed-
batch fermentation on flour hydrolysate using co-culture strategy
(Table 7). In this study, bottleneck was defined as conversion of lactate
to propionic acid. Therefore, two-step fermentation technology was
preferred in our study. Despite unsterile conditions in the batch fer­
mentations, Sabra et al. reported that there was no contamination.
Kagliwal et al. reported that, 37.70 g/L propionic acid was obtained
after 115 h using flour hydrolysate as a substrate. In this study, glucose
was obtained from hydrolysing of wheat flour starch by using amylases
to generate substrate for P. acidipropionici. However, P. freudenreichii
cannot ferment glucose to propionic acid; we performed hydrolysis of
flour for lactic acid bacteria for the first stage of propionic acid
fermentation.
In this study, contamination has been observed in long-term
fermentation when the flour hydrolysate was used. However, as long
as there is no contamination, repeated batch fermentations enable
product removal while allowing partial reuse of biomass, together with
improving cell adaptation to high product titers over long term opera­
tion. Therefore, in this two-step system substrate inhibition was reduced
and relatively higher product titres were achieved.
4. Conclusions
In this study, two stage-repeated batch propionic acid fermentations
were developed on two natural feedstocks namely whey and flour. Lactic
acid was produced in the first stage using L. plantarum and this lactic
acid was converted mainly to propionic acid using P. freudenreichii in
repeated batch fermentations. Lactic acid and propionic acid pro­
ductivities and yields were relatively higher on whey than flour. No
contamination was observed on whey in both cultures whereas propi­
onic acid fermentation was contaminated despite the sterilization of
flour hydrolysate, which was observed by gradual decline of lactic acid
yield and appearance of bacteria and yeast contamination and alcohol
measurements. Repeated batch fermentation improves the pro­
ductivities and eliminates turnaround time in single batch fermentations
allowing repeated use of part of the biomass in the fermenter. Using food
grade substrate such as whey and flour provides an advantage to supply
the product for food industry. It is known that the major limitations in
microbial propionic acid production are the slow growth rates, low
biomass yields and acid stress tolerance levels of the microorganisms
used in the process. Therefore, developing long term processes such as
repeated batch or continuous fermentations integrated with novel cell
retention and product removal techniques may alleviate such short­
comings. In addition, further work also needed to control contamination
levels also especially allowing to work with non-conventional bio-based
feedstocks. Further improvements in strains combined with new process
techniques are required for a cost-effective production of bio-based
propionic acid or similar products to be used in the food industry to
replace fossil-based propionic acid.
Funding
This research has been internally funded by Pak Gıda Üretim ve
Pazarlama A.Ş.

8
E. Bezirci et al.
CRediT authorship contribution statement
Emine Bezirci: Investigation, Writing - Original draft preparartion.
Hatice Taşpınar-Demir: Investigation, Writing - Original draft prepa­
ration. Burcu Turanlı-Yıldız: Investigation, Writing - Original draft
preparation. Atacan Erdem: Investigation. Filiz Alemdar: Supervision.
Mustafa Türker: Management and coordination, supervision, Writing -
Review and Editing.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Data availability
No data was used for the research described in the article.
Acknowledgements
We are grateful to Tom Jondiko of Pak North America (Pasadena,
USA) for reading and constructive comments.
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