104

Biotechnol. h g . 1992, 8, 104-110

A Novel Extractive Fermentation Process for Propionic Acid

Production from Whey Lactose
Vivian P. Lewis and Shang-Tian Yang*
Department of Chemical Engineering, The Ohio State University, 140 West 19th Avenue, Columbus, Ohio 43210

An extractive fermentation process was developed to produce propionate from lactose.

The bacterium Propionibacterium acidipropionici was immobilized in a spiral wound,
fibrous matrix packed in the reactor. Propionic acid is the major product from lactose
fermentation, with acetic acid and carbon dioxide as byproducts. Propionic acid is a
strong inhibitor to this fermentation. A tertiary amine was used to selectively extract
propionic acid from the bioreactor, hence enhancing reactor productivity by over 100%.
We also speculate that by selectively extracting propionic acid, lactose metabolism can
be directed to yield more propionate and less byproducts. Other advantages of extractive
fermentation include better pH control (by removing acid products) and a purer product.
The propionic acid present in the extractant can be easily stripped with small amounts
of an alkaline solution, resulting in a concentrated propionate salt. The extractant was
also regenerated in this stripping step. Thus, the process is energy-efficient and
economically attractive.

Introduction
Propionic acid is an important mold inhibitor. Its
calcium, sodium, and potassium salts are widely used as
food and feed preservatives. To date, commercial production of propionic acid is almost entirely by petrochemical routes (Playne, 1985). However, there has been
increasinginterest in producing propionic acid from whey
lactose and other cheap biomass using propionibacteria
(Blanc & Goma, 1987a; Bodie et al., 1987; Border et al.,
1987;Boyaval and Corre, 1987;Cavin et al., 1985;Clausen
and Gaddy, 1981; Crespo et al., 1990; Datta, 1981; Emde
and Schink, 1990; Hendricks et al., 1985; Hsu and Yang,
1991; Lewis, 1991; OBrien et al., 1990).
Propionic acid bacteria have long been used in the dairy
industry. These bacteria play important roles in the
development of the characteristic flavor and eye production in Swiss-type cheeses. Propionibacteria are Grampositive, nonspore-forming,rod-shaped, facultative anaerobes. The optimum pH range for growth is between 6 and
7, and at pH C 4.5 there is practically no growth (Hsu &
Yang, 1991). Like most organic acid fermentations, the
propionic acid fermentation is inhibited by acidic pHs
and the major fermentation product, propionic acid (Blanc
and Goma, 1987b; Ibragimova et al., 1969; Neronova et
al., 1967). The conventional fermentation technology for
propionate production is thus limited by low fermentation
rate and low product concentration. Furthermore, the
fermentation is heterogeneous; i.e., propionate is produced
along with other byproducts. This not only results in a
low product yield but also renders product purification
difficult and expensive. Consequently, the conventional
fermentation route for propionic acid production is
inefficient and it competes with difficulty with petrochemical routes. Presently, only small amounts of propionate are produced by fermentation of whey and are
used as a natural product in foods for the labeling purpose.
In order to make the fermentation route economically
viable, it is necessary to develop novel fermentation
processes that use highly efficient bioreactors and separations techniques.

* Corresponding author.
8756-7938/92/3008-0104$03.00/0

Recently, integrated fermentation-separation systems

have been successfully used to reduce end-product inhibition and, thus, to improve the overall process efficiency
(Daugulis, 1988; Roffler et al., 1984). To date, the most
studied process has been the extractive fermentation for
ethanol production. Several extractive fermentation systems also have been studied for organic acid production
(Bar and Gainer, 1987; Yabannavar and Wang, 1991).
Extractive fermentation removes the inhibitory, acidic
product from the reactor and, therefore, provides better
pH control on the reactor and results in higher reaction
rates. Also, products are present in relatively pure and
concentrated forms. Thus, savings in the downstream
recovery and purification costs can be realized. However,
all prior extractive fermentation studies dealt with homofermentative products, such as lactic, acetic, and citric
acids. To our knowledge, no extractive fermentation has
been studied for heterofermentative products such as propionic acid.
Successful development of an extractive fermentation
process requires careful selection of a highly efficient and
nontoxic solvent for extraction. Conventional solvents
such as alcohols, ketones, ethers, and aliphatic hydrocarbons are not efficient when applied to dilute, carboxylic
acid solutions because of the low aqueous activity of carboxylic acids resulting in low distribution coefficients
(Kertes and King, 1986). Furthermore, most organic
solvents are toxic to bacteria; they will either inhibit or
stop bacterial growth. Extractive fermentation using
conventional extractants usually suffers from solvent
toxicity and a low separation factor, and it may require
formidably expensivesolvent regeneration procedures. To
date, no extractive fermentation has been developed for
industrial applications.
Recently, several aliphatic amines have been used
successfullyto extract carboxylicacids (Wardell and King,
1978; Ricker et al., 1980; Kertes and King, 1986; Wennersten, 1983;Tamada and King, 1990;Yang et al., 1991).
The strong amine interactions with the acid allow for
formation of acid-amine complexes and thus provide for
high distribution coefficients. In addition, the high affinity
of the organic base for the acid gives selectivity for the

0 1992 American Chemical Society and American Institute of Chemical Engineers

105

Biotechnol. Rog., 1992, Vol. 8, No. 2

acid over nonacidic components in the mixture. A tertiary

amine, Alamine 336, has been shown to pose only slight
toxicity to immobilized homolactic bacteria and was used
successfully in extractive fermentation to improve the
lactic acid production rate (Yabannavarand Wang, 1991b).
In this work, we used an Alamine 336/2-octanol mixture
as the extractant in an extractive fermentation process
for propionate production from whey lactose. This extractant has higher distribution coefficients for propionic
acid than for acetic acid (Yang et al., 1991). Thus, propionic acid is preferentially extracted from the mixed acid
products present in the fermentation broth. By properly
designing the extractor, it is possible to selectively remove
propionic acid and thus to produce a pure product, even
though the fermentation is heterogeneous. We also
speculated that by selectively extracting propionic acid,
lactose metabolism can be directed toward propionic acid,
instead of acetic acid or other byproducts, to yield more
propionate. The results from a prototype laboratory unit
are reported in this article. Effects of pH, product
inhibition, and solvent toxicity on propionic acid fermentation were also studied and are discussed in this paper.

Materials and Methods

Culture and Media. Propionibacterium acidipropionici ATCC 4875 was used in this work. A synthetic lactose
medium was used as the feed to the bioreactor. This
medium contained the following (per liter): 10 g of yeast
extract (Difco), 5 g of Trypticase (BBL), 0.25 g of KzHP04,0.05 g of MnS04, and 16g of lactose. The medium
was prepared in two parts: the basal medium (without
lactose) and a concentrated lactose solution. These two
solutions were adjusted to pH 7.0 by the addition of NaOH
solution. They were then mixed aseptically, after heat
sterilization at 121 "C and 15 psig for 20 min.
In some kinetic studies, lactate was used to replace
lactose as the carbon source for cell growth. To study the
pH effect, the medium pH was adjusted by the addition
of 4 N HC1 or 4 N NaOH. For a study of the effects of
acetic and propionic acids, the media also contained various
amounts of propionic acid (0-20 g/L) or acetic acid (0-9
g/L). For a study of the solvent toxicity, the media were
prepared from distilled water with different degrees of
solvent saturation. All media were purged with N2 before
being autoclaved.
Fermentation Kinetics. Inhibition kinetics were
studied to evaluate the effects of pH, propionic and acetic
acids, and solvent toxicity on cell growth. Unless otherwise
noted, these kinetic studies were carried out at 30 "C, in
18-mL serum tubes, and lactate was used as the carbon
source in the growth medium to minimize pH change due
to growth. (About 1 mol of propionic and acetic acids
would form from each mol of lactate fermented.) Each
tube containing 10mL of the medium was inoculated with
0.5 mL of 1-day-old culture. Cell growth was monitored
by direct measurement of the optical density a t 660 nm
(ODm) in the serum tube using a spectrophotometer (Sequoia-Turner, Model 340). The specific growth rate (p)
was determined from the slope of the semilogarithmicplot
of OD versus time. Only the initial growth rate data were
used to evaluate the effects of pH, acetate, propionate,
and solvent. All experiments were at least duplicated to
get consistent and statistically meaningful results.
Extraction. The amine extractant, Alamine 336 (from
Henkel Corp.), was mixed with a diluent, 2-octanol, at
various proportions to determine the optimal extractant
composition for propionic and acetic acids extraction. Acid
solutions (pH 3.0) containing 30 g/L propionic or acetic
acid or both were mixed with an equal volume of the exN

rec cle

out

Feed in

Bioreactor

Figure 1. The immobilized cell bioreactor and extractor used

in ex situ extractive fermentation.

tractant. After equilibrium was reached at 30 "C, phase

separation was facilitated by centrifugation. The distribution coefficient at equilibrium was determined from
the acid concentrations in the organic and aqueous phases.
Organic acids in the aqueous phase were determined by
high-performance liquid chromatography (HPLC) or gas
chromatography (GC). The organic phase was backextracted with an alkaline solution first, and then, this
solution was analyzed to determine the amounts of acids
present in the extractant (organic phase). A material
balance was performed to check that the total amounts of
the organic acids before and after extraction were equal.
Immobilized Cell Bioreactor. The bioreactor used
for extractive fermentation was made of a 2-in. glass column
packed with spiral wound, cotton towel as the matrix for
cell immobilization. This packing design allows good
contact between two different moving phases (such as gasliquid or solvent-aqueous two phases). The reactor
packing length was 12 in., and the reactor working volume
was 600 mL. These reactors were autoclaved twice and
then filled with sterilized growth medium and inoculated
with 50 mL of cells. The cells in the bioreactor were allowed
to grow for 2-3 days before a continuous feed was started.
These reactors were fed with a lactate medium for several
weeks to build up the cell mass in the reactor. The lactate
medium was used to avoid a significant pH drop in the
reactor. After the reactor packing has been saturated with
the cells, the reactor feed was switched to the lactose
medium.
Extractive Fermentation. Extractive fermentation
was studied first with external extraction and then with
in situ extraction. The experimental apparatus used for
the ex situ extraction process is shown in Figure 1. The
extraction column was made of a 1-in.glass column packed
with 0.2541. ceramic saddles. The packing length was 18
in. Both the bioreactor and the extractor were controlled
at 30 "C. The extractant used was 40% (w/w) Alamine
336 in 2-octanol. Before use, fresh extractant was contacted with the medium to wash off any water-soluble
impurities in the solvent and to saturate the solvent with
the ingredients present in the medium. After each
extraction use, the extractant was regenerated by contacting it with 1N NaOH solution to strip propionic and
acetic acids from the solvent. The regenerated extractant
was then reused for the next extraction.
The extractive fermentation was operated at the following conditions: feed rate, 0.3 L/d; dilution rate, 0.5
d-l; recycle rate, 1.2 L/d;extractant flow rate, 1.68L/d. At
appropriate time intervals, samples were collected from
the bioreactor outlet and two extractor outlet streams
(aqueous and organic phases). For the in situ extraction
process, the extractant was fed directly into the reactor

Biotechnol. Prog,, 1992, Vol. 8, No. 2

106

o.201

O.1
0.05

0.25

Eul
cn

0.15&

pH 5.45

pH 5.9

0.15

i
t

0.0ot.
4

'

'

' .

'

"

'

"

' 1
7

PH
Figure 2. Effect of pH on the specific growth rate.

from the reactor bottom. All other operating conditions

remained unchanged except that there was no recirculation. Both the aqueous and organic phases were collected
from the outlet at the top of the reactor. All aqueous
samples were analyzed for pH and cell concentration
immediately. These samples were then stored in a freezer
for future HPLC analysis. Organic-phase samples were
back-extracted with 0.33 N NaOH, and the resulting
aqueous samples were also stored for future HPLC
analysis.
Assay Methods. A high-performanceliquid chromatograph was used in analyzing organic compounds including
lactose and propionic and acetic acids in the fermentation
broth. A Waters HPLC system (6000Apump,710B WISP
injector, 481 variable wavelength detector, and R410 refractometer) and a Bio-Rad HPX-87H column (at 46 "C)
were used; 0.013 N HzS04, a t a flow rate of 0.6 mL/min,
was used as the eluant. Samples containing low concentrations of volatile compounds (e.g., propionic and acetic
acids) were determined using a gas chromatograph (GC)
equipped with a flame ionization detector (FID) and a
fused silica megabore column (DBWAX, 15 m X 0.534
mm; J & W Scientific). The carrier gas was N2 at a flow
rate of 30 mL/min. The operating temperatures were
injector, 110 "C; column, 110 "C; and detector, 160 "C.
Ethanol or butyric acid was used as an internal standard
in determining the acid concentration in the sample.
Suspended cell growth was monitored by the optical
density (OD) at 660 nm using a spectrophotometer. OD
was measured in a 1.5-mL polystyrene cuvette with a light
path of 10 mm. Immobilized cell growth in the fibrous
matrix was examined by using a scanning electronic
microscope (SEM).Detailed procedures can be found
elsewhere (Lewis, 1991).

Rssults
Fermentation Kinetics. Effect of pH. Figure 2
shows the effect of pH on the specific growth rate ( p ) .
When lactose was the growth substrate, this propionibacterium has an optimum pH at -7 and p decreases
with a decreasing pH. Note that p dropped drastically a t
pH below 5.
Effect ofPropionic Acid. Propionic acid is known to
inhibit the propionic acid fermentation. Figure 3 shows
that at all pH values studied, p dropped dramatically with
an increasing propionate concentration. However, this
decreasing trend leveled off when the propionate concentration was greater than -10 g/L.
Effect of Acetic Acid. At pH 6.15, acetic acid did not
affect p significantly (Figure 4). However, as also shown
in Figure 4, slight acetate inhibition was found a t a lower
pH value, but the effect was not as dramatic as that found
with propionate.
Solvent Toxicity. It is known that the organic solvent
used for extraction usually imposes some degree of toxicity

15

10

25

20

30

Propionic acid (g/L)

Figure 3. Effect of propionic acid on the specific growth rate.

0.151

. , .

pH 6.15
'C

CI

0.10

pH 5.60

0.05 -A-

&-A
A-A

ol

10

12

Acetic acid (g/L)

Figure 4. Effect of acetic acid on the specific growth rate.

0.31,.
,

7
;

, .

I . .

1 . 1

pH 7.0

d
v)

0.0

20

40

60

80

100

SOLVENT SATURATION , %

Figure 5. Effect of solvent concentration on the specific growth

rate.

to the microorganism. The toxicity of the extractant

(Alamine 336/2-octanol mixture) dissolved in the growth
medium was studied first with batch cultures of suspended
cells. As shown in Figure 5, the extractant inhibits the
cell growth, especially at concentrations higher than 60 %
saturation. However, even at 100% saturation, the
bacterium still has significant growth.
The solvent toxicity was also tested with immobilized
cells in the continuous process. The reactor was operated
under plug-flow conditions and fed with media containing
-45 g/L lactate and solvent at different degrees of
saturation. Steady-state concentrations of lactate, propionate, and acetate in the effluent were monitored and
are shown in Table I. As shown in this table, generally
there were more substrate left and less products produced
when more solvent was present, indicating that fermentation was inhibited by the solvent. Nevertheless, even
at 1005% saturation the solvent toxicity on the fermentation
was not severe. As also shown in Table I, the Ypand Y,
values remained almost unchanged at all levels of solvent
saturation. Thus, the presence of solvent did not affect
product yields from the lactate fermented, although the
total acid production could be lower at higher solvent
concentrations due to slower reaction rates.

Blotechnol. prog., 1992, Vol. 8, No. 2

107

Table I. Effects of Solvent Toxicity on Propionic Acid

Fermentation
Ypn Y,"
solvent concn
lactic
propionic
acetic
(% saturation) acid (g/L) acid (g/L) acid (g/L) (g/g) (g/g)
0.68 0.22
6.13
16.75
19.33
0
5.96
0.67 0.22
18.40
25
17.62
0.62 0.20
17.34
5.47
50
17.19
0.73 0.23
13.83
4.32
75
26.00
0.69 0.22
15.08
4.84
100
23.00
Ypis propionic acid yield, and Y. is acetic acid yield; both are
based on the amount of lactate fermented.

.-a

(I

0.25

0.50

0.75

1 .oo

Fraction of reactor length

Figure 7. Propionic acid and pH profiles in the bioreactor

without extraction. The reactor was operated under plug-flow
conditions, and samples were taken from the reactor at different
reactor lengths.

10
U

20

10

30

Alamine

acetic acid

'

40

a
50

(%)

/31

,do

10

20

Alamine

30

40

50

40

50

(%)

10

20

Alamine

30
(%)

Figure 6. Effect of Alamine concentration on distribution

coefficients: (a) propionic acid; (b) acetic acid, and (c) mixture
of propionic and acetic acids.

Extraction Kinetics. It is known that the diluent 2octanol can greatly increase the extracting power of the
amine extractant by providing more solvating ability to
the solvent (Yang et al., 1991). The optimal composition
of the Alamine/2-octanol mixture was studied first to
obtain the highest distribution coefficient (Kd) for propionic acid. Figure 6 shows that the optimal wt % of
Alamine 336 in 2-octanol was -40%, where Kd reached
maximum values for both propionic and acetic acids. The
Kd values were about the same for extraction with propionic or acetic acid alone (Figure 6a,b) or in a mixture
(Figure 6c). However, the Kd values for propionic acid

are much higher than those for acetic acid, suggesting that
this extractant can extract propionic acid better. The
amine extractants generally extract much better with
longer carboxylicacids with large hydrophobic alkyl group
(Yang et al., 1991).
Extractive Fermentation. When lactose was the
carbon source in continuous, immobilized cell fermentations, the reactor performance deteriorated with time if
no pH control was provided. After running for a week,
only a small amount of lactose was fermented. This was
attributed to low pH and propionic acid inhibition. As
shown in Figure 7,when the reactor was operated under
plug-flow conditions, the reactor pH dropped quickly with
propionic acid production and it almost reached the lower
pH limit at the first 25% of the reactor length.
Ex Situ Extraction. Ex situ extraction of propionic
acid with amine extractant was performed to demonstrate
the advantages of extractive fermentation over conventional fermentations. As shown in Figure 8a, with extraction, the lactose concentration in the bioreactor outlet
stream decreased from 11g/L to -6 g/L, indicating that
more lactose was fermented under extractive fermentation
conditions. This improvement was due to the reduced
level of propionic acid and a proper pH value maintained
in the bioreactor. As shown in Figure 8b, the concentration
of propionic acid in the bioreactor reduced from 3.3 g/L
to -2.0 g/L. The propionic acid concentration in the
recycle stream after extraction was less than 1 g/L, and
significant amounts of propionic acid produced were
extracted into the organic phase. The concentrations of
acetic acid in various streams are shown in Figure 8c. It
is clear that the solvent extraction only removed a small
portion of the acetic acid produced and that most acetic
acid remained in the recycle stream.
The total propionic acid production in terms of grams
per liter can be calculated by doing the material balance
either around the bioreactor using the two aqueous-phase
propionic acid concentrations as given by
pa(g/L) = + 4(p1 - p 2 )
or around the system using the two outlet concentrations
of propionic acid (aqueous and organic phases) as given
by

Pb(g/L) = PI+ 5.6P3

PI,Pz,
and P3 denote propionic acid concentrations in the
reactor outlet, recycling stream entering the reactor, and
organic-phase outlet from the extractor, respectively. In
the above two equations, the number 4 is the ratio between
the recycle rate and feed rate, and 5.6 is the ratio between
the extractant flow rate and the feed rate. Acetic acid
production was also calculated similarly.

Blotechnol. hog., 1992, Vol. 8, No. 2

2
.
-

12

'e

'
2
0

.-0C

Pa
Pb

Ab

;io'

'

20

40

SO

60

Time

-0-0-

100

120

.
.

(hours)

P1
A-A-A

20

80

60

40

Time

100

120

(hours)

,CI

2a

d,

'

'210'

'

'410'

'

'610' ' '810'

Operating time
A1

en

P-

'{
.a

n-

0.6

0.3

-a

0.0

a-

ne

A 3 -0

0-0-0-

'

io

(hours)

Figure 9. Performance of the ex situ extractive fermentation:

(a) overall propionic acid production; (b) overall acetic acid
production.

I
0

20

40

Time

60

80

100

120

(hours)

Figure 8. Performance of the ex situ extractive fermentation:

(a) the concentration of lactose in the reactor outlet; (b)
concentrationsof propionic acid in the reactor outlet (Pl),in the
aqueous phase of the extractor outlet (P2), and in the organic
phase of the extractor outlet (P3); (c) concentrations of acetic
acid in the reactor outlet (Al), in the aqueous phase of the
extractor outlet (A2), and in the organic phase of the extractor
outlet (A3).

As shown in Figure 9, both calculation methods gave

about the same result. Without extraction, propionic acid
production was only -3 g/L, and with extraction it
increased to -7 g/L (Figure 9a). Similarly, acetic acid
production increased from 0.9 g/L (without extraction) to
- 2 g/L (Figure 9b). Hence, with extraction, both propionic acid and acetic acid production increased by more
than 100%. The weight ratio of propionic acid to acetic
acid (PIA)remained unchanged a t -3.3 with or without
extraction.
On the basis of the amount of lactose fermented, the
propionic and acetic acid yields from the extractive
fermentation were 0.73 (g/g) and 0.22 (g/g), respectively.
These yield values were significantly higher than those
previouslyfound for batch fermentations with lactose (Hsu
and Yang, 1991). Also, on the basis of the balanced dicarboxylic acid pathway (Playne, 1985), the theoretical
yield values are 0.55 (gig) for propionic acid and 0.22 (gig)
for acetic acid, and the PIA ratio is -2.5.
One advantage of using extractive fermentation for propionic acid production is to obtain a purer product. As
shown in Figure 10, the propionic acid to acetic acid ratio
(PIA)in the extractant was -4.0, and the PIA ratio in the

* n e-

e-

e-

* L e Aq.-after

a
0

25

50

Time

75

100

125

150

(hours)

Figure 10. Propionic to acetic acid ratio in various streams from

the ex situ extractive fermentation.

aqueous phase was 2.0 before extraction and 1.2 after

extraction. Hence, propionic acid was preferentially
recovered and purified to some degree from the product
mixture by the extraction. This separation and purification effect can be further enhanced by optimizing the
design and operating conditions of the extractor.
In Situ Extraction. Fermentation with in situ extraction was also studied, and the results are shown in
Figure 11. As shown in this figure, the lactose concentration in the outlet stream decreased with time from 11
g/L to -4.5 g/L. However, the total propionic acid
production increased only initially; it then decreased to
a lower level (Figure 12). At the same time, both glucose
and galactose increased from 0 to -3.6 g/L (Figure 11).
Glucose and galactose were not found in previous fermentation experiments with ex situ extraction or no
extraction. This indicates that the direct contact of the
solvent with the cells might have caused some cells to lyse
and release 8-galactosidase. Consequently, the reactor
productivity was low. However, the reactor still maintained production, indicating that there were viable cells

109

Biotechnoi. PTOg., 1992, Vol. 8, NO. 2

0
0

i
cn

Lactose
Glucose

+ Galactose

"0

50

100

150

200

250

300

350

Time (hours)

using solvents that are essentially insoluble in water. The

solubility of Alamine 336 in water is less than 5 ppm.
In this work, the dissolved solvent did not pose any
major problem to the fermentation, especially for the case
of ex situ extraction. It is noted that, with a recycle ratio
of 4, the solvent concentration in the bioreactor should be
below 80% saturation. On the contrary, phase toxicity
seemed to be the major cause of decreased propionic acid
production from the in situ extractive fermentation. Thus,
cells should be protected from directly contacting the
solvent to allow the in situ extractive fermentation to work.
Effects of pH. For extractive fermentation, the reactor
performance is highly dependent on the pH. Both the
fermentation and extraction are highly pH-dependent. The
distribution coefficient, &, is greatly affected by the
solution pH (Yanget al., 1991). It is known that the amine
extractant only extracts the undissociated acids. The
distribution coefficient & is essentially zero at pH 7.0. It
increases with a decrease in the pH value and reaches its
maximum value at pH 4.0 (Yang et al., 1991). On the
other hand, cells grow better at pH values higher than 5.0,
with an optimum around pH 7. Thus, a higher pH favors
the fermentation, whereas a lower pH favors the extraction.
Therefore, it is essential to optimize the reactor pH profile
to facilitate both fermentation and extraction.
Without extraction, the pH value of the outlet stream
from the bioreactor was -4.3, which is close to the pH
limit for cell growth. With ex situ extraction, this pH
value increased to -4.8. The outlet stream from the
extractor had a pH value of -6.0. The & values reported
in Figure 6 were obtained at an equilibrium pH of less
than 4.0. Since Kd decreases with an increasing pH, the
Kd values in the extractive fermentation would be lower
than those shown in Figure 6.
It is noted here that the conditions chosen to operate
the bioreactor in this study were not anywhere near the
optimal conditions for these bioreactors either with or
without extraction. The objective of this study was to
demonstrate the concept and advantages of extractive
fermentation for propionic acid production. For practical
purpose, the lactose concentration in the feed stream
should be -5% and the residual lactose concentration in
the effluent stream should be close to zero to minimize
waste disposal problems. Further work is needed to
optimize the reactor and extractor before a process can be
developed for industrial uses.
Extractive Fermentation Process. An extractive
separation and recovery process normally involves two
steps: extraction and solvent regeneration. The amine
extractant only extracts undissociated acid and does not
extract organic acids at basic conditions. This allows
solvent regeneration through back-extraction with an
alkaline solution. Therefore, the extractant can be easily
regenerated by strippingwith a small volume of an alkaline
solution. In the mean time, a concentrated organic salt
solution is obtained. This allows efficient propionate salt
production by using an extractive fermentation process.
Figure 13 shows a conceptual process flowsheet for propionate production from whey permeate.
Our preliminary economic analysis indicates that for a
typical dairy plant processing 1000 000 lbs of whey per
day, about 40 000 lbs of calcium propionate can be
produced at a cost of --$0.15/lb. The cost analysis was
based on 95 7% conversion of lactose a t a retention time of
24 h, which have been found to be adequate for the bioreactor fed with 4 % lactate without the effects of extraction
(Lewis, 1991). With the effects of extraction,a better bioreactor performance may be realized. The extractive
fermentation process for propionate production is thus

cn

20
m

50

100

150

200

250

300

350

Time (hours)

Figure 11. Performance of the in situ extractive fermentation:

(a) outlet concentrations of lactose, glucose, and galactose; (b)
outlet concentrations of propionic acid in the aqueous phase (Pl)
and in the organic phase (P3).

Time (hours)

Figure 12. Overall propionic acid production from the in situ

extractive fermentation.

present in the reactor. These cells probably were inside

the fibrous matrix and thus were protected from the
solvent.

Discussion
Solvent Toxicity. Solvent toxicity can exert on the
microorganisms at both the molecular level and the phase
level. Toxicity at the phase level comes from the direct
contact of the solvent phase with the cells, which may
block nutrient diffusion from the medium to cells due to
solvent coating and may disrupt the cell wall due to
increased surface tension. Toxicity at the molecular level
comes from the dissolved organic solvent which can inhibit
enzymes or modify cell membrane permeability. Phase
toxicity can be eliminated by using hydrophobic membranes (Cho and Shuler, 1986)or cell immobilization (Yabannavar and Wang, 1991a). Solvent toxicity at the
molecular level can be reduced to a minimal degree by

110

Biotechnol. Prog., 1992, Vol. 8, No. 2

Fermentallon

Solvent
Exlracllon

IiI

40,000 I b s
Ca-Propionate

1,GOG,OOO l b s
Whey P e r m e a t e
5< L a c t o s e

Recycle 7

L i m e (CaO)

Solution

Figure 13. A conceptual extractive fermentation process for

propionate production from whey lactose.

economically attractive. However, further research and

development are necessary to optimize and scale-up the
process.

Conclusion
The feasibility and advantages of using the extractive
fermentation process for propionic acid production from
lactose have been demonstrated in this work. The mixture
of Alamine 336/2-octanol has a high extraction coefficient,
is generally not toxic to the propionic bacterium, and can
be used for ex situ extraction during propionic acid
fermentation. Over a 100% increase in reactor productivity was attained. The improved reactor productivity
was attributed to proper pH control and reduced product
inhibition through the removal of propionic acid by solvent
extraction. Propionic acid yields were enhanced, and a
purer product can be obtained by using the extractive
fermentation process.

Acknowledgment
This work was supported in part by the Ohio Department of Transportation and the U.S. Department of
Energy Innovative Concept Program. The financial
support to S.-T.Y. from the Du Pont Young Faculty Award
is also gratefully acknowledged.

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Accepted January 6,1992.
Registry No. Propionic acid, 79-09-4;lactose, 63-42-3.