Appl Microbiol Biotechnol (2012) 93:10571063
DOI 10.1007/s00253-011-3595-6
BIOTECHNOLOGICAL PRODUCTS AND PROCESS ENGINEERING
High-level production of 1,3-propanediol from crude glycerol
by Clostridium butyricum AKR102a
Erik Wilkens & Anne Katrin Ringel & Diana Hortig &
Thomas Willke & Klaus-Dieter Vorlop
Received: 15 August 2011 / Revised: 9 September 2011 / Accepted: 18 September 2011 / Published online: 5 October 2011
# Springer-Verlag 2011
Abstract The aim of this study was to optimize a biotech-
nological process for the production of 1,3-propanediol (1,3-
PD) based on low-quality crude glycerol derived from
biodiesel production. Clostridium butyricum AKR102a was
used in fed-batch fermentations in 1-L and 200-L scale. The
newly discovered strain is characterized by rapid growth,
high product tolerance, and the ability to use crude glycerol
at the lowest purity directly gained from a biodiesel plant
side stream. Using pure glycerol, the strain AKR102 reached
93.7 g/L 1,3-PD with an overall productivity of 3.3 g/(L*h).
With crude glycerol under the same conditions, 76.2 g/L 1,3-
PD was produced with a productivity of 2.3 g/(L*h). These
are among the best results published so far for natural
producers. The scale up to 200 L was possible. Due to the
simpler process design, only 61.5 g/L 1,3-PD could be
reached with a productivity of 2.1 g/(L*h).
Keywords Fermentation . Crude glycerol .
1,3-Propanediol . Clostridium butyricum
Introduction
1,3-Propanediol (1,3-PD) has a wide range of potential
applications. Of these, the use in polymerization reactions
is of increased interest. A commercially available poly-
E. Wilkens : A. K. Ringel : D. Hortig : T. Willke (*) :
K.-D. Vorlop
Johann Heinrich von Thnen-Institut (vTI),
Institute of Agricultural Technology
and Biosystems Engineering (AB),
Bundesallee 50,
38116 Braunschweig, Germany
e-mail: thomas.willke@vti.bund.de
trimethylane terephthalate is sold as Sorona (DuPont) or
Corterra (Shell) and can, for example, be used for carpets
or textiles. Other than the usage as a building block for
chemical reactions, 1,3-propanediol can be used as coolant/
antifreeze for motors or fuel cells (Eaton et al. 2004) or to
improve the properties of solvents, lubricants, laminates,
and adhesives (Zeng and Biebl 2002). Its low potential for
skin irritation (Belcher et al. 2010) also makes it interesting
for applications in cosmetics, where it can serve as a
humectant or emollient.
One way to produce 1,3-PD is by means of chemical
synthesis. Two major routes are the conversion of acrolein
to 1,3-PD via 3-hydroxypropionaldehyde (Degussa/DuPont
process) or the conversion of ethylene oxide to 1,3-PD via
3-hydroxypropionaldehyde (Shell process) (Kraus 2008).
Disadvantages of these production routes are toxic inter-
mediates or high equipment costs (Liu et al. 2010) and the
dependence on feedstock based on fossil resources. Those
difficulties can be avoided by applying microbial conver-
sion of renewable resources to produce 1,3-PD.
A wide variety of natural organisms are known
which convert glycerol (Gly) to 1,3-PD via 3-
hydroxypropionaldehyde. Enterobacteriaceae such as
Klebsiella pneumoniae (Homann et al. 1990), Citrobacter
freundii (Boenigk et al. 1993), and Enterobacter agglomerans
(Barbirato et al. 1998); Clostridiaceae such as Clostridium
butyricum (Biebl et al. 1992); or Lactobacillaceae such as
Lactobacillus brevis (Schuetz and Radler 1984) have been
reported to produce 1,3-PD under anaerobic conditions.
Strains of K. pneumoniae are also capable of producing 1,3-
PD under aerobic or micro-aerobic conditions (Cheng et al.
2004; Liu et al. 2007). On other occasions, natural producers
were mutated or genetically modified to improve the existing
capabilities (Otte et al. 2009) or eliminate by-products (Xu et
al. 2009). Additionally, a different kind of genetically
1058
engineered organism has been put to use. DuPont is
commercially converting glucose from cornstarch to 1,3-PD
(Zemea) using a specifically genetically engineered strain of
Escherichia coli (Nakamura and Whited 2003).
Glycerol is a main by-product of fat saponification or the
transesterification process during biodiesel production. For
every ton of biodiesel, 100 kg of glycerol is produced
(Yazdani and Gonzalez 2007) which can either be used
directly or further refined up to pharmaceutical grade.
The production costs of the microbial conversion have to
be reduced in order to compete with conventional chemical
synthesis. Raw materials for the fermentation medium and
glycerol are the main cost drivers and account for more
than 50% of the total costs (Hirschmann et al. 2005). Since
crude glycerol (CG) does not require multiple expensive
purification steps, it can be obtained at significantly lower
prices than pure glycerol (PG). Furthermore, the rising
production of bio-based fuels will contribute to the
availability of crude glycerol as an abundant and econom-
ically priced carbon source. Thus, its usage as feedstock
presents an effective method for a significant cost reduction
of the process. It has to be considered though that
potentially present impurities such as methanol, fatty acids,
soap, heavy metals, or salt can prove to be inhibitory
(Petitdemange et al. 1995; Chatzifragkou et al. 2010). It has
been shown that the influence on 1,3-propanediol produc-
tion can differ noticeably depending on the source and
producer of the used glycerol and the applied strain (Moon
et al. 2010). Thus, a strain has to be found that is not, or is
only, slightly affected by a variety of impurities, or an
applicable pretreatment method has to be developed.
Methods such as washing with a suitable alcohol (Rehman
et al. 2008) or acid treatment (Moon et al. 2010) have been
published.
Materials and methods
Microorganism
The bacterial strain AKR102a used in this study was
isolated from soil samples at our institute (Ringel et al.
2011) and has been identified as C. butyricum based on
partial 16S rRNA and fatty acid analyses. The identification
was performed at the Collection of Microorganisms and
Cell Cultures (DSMZ) where the strain was also deposited
(collection number DSM 25047). For experimental usage,
the strain was stored in 50% pure glycerol at 80C. Prior
to the start of fermentation, at least three generations of
precultures were grown at 32C as still cultures in 50-mL
glass vials sealed with butyl rubber septums filled under
anaerobic conditions with 30 mL mineral salt medium
(see below).
Appl Microbiol Biotechnol (2012) 93:10571063
Media and solutions
All of the used media components, with the exception of crude
glycerol, were p.a. quality and purchased from either Merck
(Germany), SigmaAldrich (USA), or Roth (Germany).
Per liter of distilled water, the preculture vial medium
(Bock 2004) contained: 9.09 g KH2PO4, 0.53 g NH4Cl,
0.123 g MgSO47H2O, 0.017 g CaSO42H2O, 0.01 g
FeSO47H2O, 1 g yeast extract, 1 mL of a 1-g/L resazurin
solution, 0.28 g L-cysteineHCl, and 2 mL trace element
solution. Per liter of distilled water, the trace element
solution (DSMZ medium 144) contained: 64 g nitrilotri-
acetic acid, 1 g FeCl34H2O, 0.5 g MnCl24H2O, 0.85 g
CoCl26H2O, 0.5 g CaCl22H2O, 0.5 g ZnCl2, 0.1 g CuCl2,
0.05 gH3BO3, 0.05 g Na2MoO42H2O, 0.13 g NiCl26H2O,
5 g NaCl, and 0.1 g Na2SeO35H2O. The pH value of the
trace element solution was adjusted to 6.5 with NaOH/HCl.
Glycerol was added to the medium as a carbon source (25 g per
liter), either as pure glycerol (98%) or crude glycerol K602
provided by Biopetrol Schwarzheide GmbH (Germany). The
crude glycerol originated from transesterification of rapeseed
oil and contained 55% (wt/wt) glycerol, 1% (wt/wt) fatty acids,
and 31,408 ppm chloride. First preliminary experiments had
shown that a simple pretreatment of the crude glycerol led to
notable performance increases (data not shown). First, a
floating layer, probably partially comprised of free fatty acids
that developed when the glycerol was stored at room
temperature for 2 days, was removed using a separating
funnel. Afterwards, the glycerol was mixed with 6.67 g/L
synthetic hydrotalcite (SigmaAldrich, USA) and stirred at
room temperature overnight. Afterwards, the hydrotalcite was
removed via centrifugation. Prior to usage, the hydrotalcite
was heated to 550C for 2 h. The pH value of the complete
mineral salt medium was adjusted to 7. Per liter of distilled
water, the medium for the fermentation experiments (Bock
2004) contained: 2.71 g KH2PO4, 2.69 g NH4Cl, 0.1857 g
MgSO47H2O, 0.025 g CaSO42H2O, 0.015 g FeSO47H2O,
5 g yeast extract, 0.714 mL of a 1-g/L resazurin solution, and
3 mL of the above-mentioned trace element solution. As
carbon source for the 1-L fermentations, either pure glycerol
or pretreated crude glycerol (see above) was added up to a
final glycerol concentration of approximately 25 g/L. Two
principal variations were conducted when using crude
glycerol, the first being the replacement of 2.69 g NH4Cl
with 4.84 g NH4SO4 due to the already high chloride
concentrations resulting from the use of hydrochloride acid
during the glycerol processing, and the second being the
change of the base used for pH control from sodium
hydroxide to ammonium hydroxide. Since sodium methylate
was used as the catalyst during the transesterification process,
the base was changed in order to reduce the input of sodium
into the fermenter when crude glycerol was used. Thus, both
changes were aimed at limiting the amount of sodium
Appl Microbiol Biotechnol (2012) 93:10571063
chloride. For the 200-L fermentation, the pretreatment of the
crude glycerol was limited to the removal of the floating
layer, and the starting concentration was reduced to 20 g/L.
The pH value of the complete mineral salt medium was
adjusted to 7.0. Approximately 1 h before inoculation, 0.3%
(v/v) of a sterile cysteine solution was added to the fermenter
to ensure optimal anaerobic conditions at the time of
inoculation. This solution contained 50 g/L L-cysteineHCl
which was diluted in oxygen-free distilled water under
anaerobic conditions and stored in 50-mL glass vials sealed
with butyl rubber septums. The feed solution for the 1-L
fermentations was composed of either 80% (wt/vol) pure
glycerol or undiluted pretreated crude glycerol. Furthermore,
40 g/L yeast extract was added to the feed solution. If needed,
the remaining volume was filled up with distilled water. The
feed solution was autoclaved for 20 min at 121C.
For the 200-L fermentation, the feed solution was made
up entirely out of unsterile crude glycerol whose floating
layer had been removed but without any further treatment
or addition of yeast extract. During fermentation, it was
stored at room temperature.
1-L scale
The small-scale fed-batch experiments were carried out in
double jacket glass reactors with a maximum working volume
of 1 L. The temperature was maintained at 32C with
circulating tempered water, and the broth was stirred at an
agitation speed of 400 rpm. pH was maintained at 6.95 by
addition of 10 M sodium hydroxide (pure glycerol) or 25%
ammonium hydroxide (crude glycerol). The addition was
triggered by the data acquisition and analysis software
DASYLab 9 (National Instruments, USA) after comparison
with a preset value. Cell-free samples were automatically
collected via filtration probes equipped with standard flow
FISP ceramic membranes (Flownamics, Inc., USA) at
regular intervals and autonomously analyzed by HPLC. The
results of glycerol and 1,3-propanediol were then compared
with predefined set points by means of DASYLab, and feed
dosage was triggered if needed, while the desired concen-
tration of glycerol declined with rising concentrations of 1,3-
propanediol. In order to provide glycerol during the sampling
cycles, the feed dosage was directly linked to the addition of
the base nearly stoichiometrically at a ratio of approximately
2.4 to 1. In addition to the automated sampling, manual
samples of 1 mL were taken for measurement of the optical
density and HPLC analysis of by-products. Anaerobic
conditions were maintained by continuous gassing with
0.6 L/h nitrogen. The reactors were inoculated with a vial
preculture which had grown to an optical density of 4.5 to 5,
resembling a cell dry weight of approximately 1 g/L. The
inoculation volume used amounted to 3% (v/v). Foaming
was inhibited by addition of the anti-foaming agent
1059
Struktol J 673 (Schill + Seilacher GmbH, Germany) if
necessary.
200-L scale
The 200-L fed-batch fermentation was carried out in a Pilot-
Fermenter Type P (Bioengineering AG, Switzerland) with a
maximum working volume of 300 L. The preculture was
carried out in a batch fermentation using a Biostat E fermenter
(Braun Biotech International GmbH, Germany) with a
maximum working volume of 15 L. When the culture had
grown to an optical density of about 5, it was anaerobically
transferred to the type P with the inoculation volume
amounting to 6% (v/v). While the temperature was main-
tained at 32C, anaerobic conditions were maintained by
continuous gassing with 30-L/h nitrogen at a stirring rate of
50 rpm. The pH was maintained at 6.9 via addition of
NH3OH. The concentration of the base had to be reduced to
10% due to technical requirements. Samples were taken at
regular intervals and analyzed by HPLC. Depending on the
results obtained, the feed dosage was then manually adjusted
to constantly provide sufficient glycerol. The final volume at
the end of fermentation amounted to approximately 200 L.
Analytical methods
Glycerol, 1,3-propanediol, butyric acid, acetic acid, and
lactic acid in the cell-free supernatant were quantified by
HPLC equipped with an Aminex HPX-87H cation-
exchange column (BioRad Laboratories, USA) using a
column temperature of 60C, 0.05 M H2SO4 as mobile
phase at a flow rate of 0.7 mL/min, a refractive index
detector (Knauer GmbH, Germany), and a UV detector
(Shimadzu Corp., Japan). Optical density was measured at
605 nm with a T80 UV/VIS Spectrophotometer (PG
Instruments Limited, UK). Frequently, observations of cell
samples were performed using a Zeiss microscope (Axioplan,
Carl Zeiss AG, Germany).
Results
1-L pure glycerol fermentation
The fermentation was started with a glycerol concentration
of 28 g/L (Fig. 1). Dropping levels of glycerol led to the
activation of the feed dosage after 5 h. The highest
productivity was obtained between the fifth and twelfth
hour of process operation at a level of about 8.1 g/(L*h).
Afterwards, productivity started to decline noticeably. The
maximum 1,3-PD concentration was reached after 28 h and
amounted to 93.7 g/L with a residual glycerol concentration
of 6.4 g/L (Fig. 1). The overall productivity can therefore
1060 Appl Microbiol Biotechnol (2012) 93:10571063

100 50
acid concentration increased throughout the whole process,
90 45
reaching 20.3 g/L after 28 h.
glycerol; 1,3-propanediol [g/L]

80 40

optical density (605 nm)

70
60
50
40
30
20
10
0 0
0 5 10
Fig. 1 1-L fed-batch fermentation of AKR102a on pure glycerol: 1,3-
propanediol concentration (open square, grams per liter), glycerol
concentration (closed circle, grams per liter), and formation of
biomass (closed star, optical density at 605 nm)
be calculated at 3.3 g/(L*h) with a production yield of
0.522 g1,3-PD/gGly or 0.632 mol1,3-PD/molGly, respectively.
Especially during the first 10 h of production, it can be seen
that the formation of 1,3-PD is growth associated. In this
context, it has to be noted that a declining optical density
does not necessarily indicate a stop of cell growth, but
merely that the rate of cell lysis is higher than continued
growth. Additionally, fermentation products might be
released from the cell upon lysis.
Production of organic acids (Fig. 2) was also clearly
noticeable with acetic acid being the first and butyric and
lactic acid following with a slight delay. Formation of acetic
acid ceased fairly early together with lactic acid with
concentrations after 28 h being 7.5 and 1.0 g/L. Butyric
22
butyric acid; acetic acid; lactic acid [g/L]
35 1-L crude glycerol fermentation
30
25 The utilization of crude glycerol in comparison to pure
20 glycerol was tested after applying minor modifications. The
glycerol level at the start of fermentation amounted to
15
26.5 g/L (Fig. 3). The segments of low glycerol levels are
10
the result of adapting the process operation to the lower
5 glycerol content of the crude glycerol. The productivity
reached its peak of 7.6 g/(L*h) between the fourth and tenth
15 20 25 30 35
hour of process operation. The value and duration are
time [h]
therefore comparable to the performance using pure
glycerol. A relatively rapid drop of productivity can be
observed after 60 g/L 1,3-PD was surpassed. The maximum
concentration of 1,3-PD after 32.5 h reached 76.2 g/L with
a residual glycerol concentration of 12.5 g/L. With the
overall productivity of 2.3 g/(L*h) being lower, the
production yields that could be achieved are equivalent to
the fermentation using pure glycerol with values of
0.514 g1,3-PD/gGly or 0.622 mol1,3-PD/molGly.
The order of formation of by-products (Fig. 4) remains
the same, as does the amount of acetic acid with a
concentration of 7.7 g/L. But a butyric acid concentration
of just 12.5 g/L and a continuous rise of lactic concen-
trations up to 6.0 g/L distinctively set it apart from the
results obtained previously with pure glycerol.
200-L fermentation
The suitability of a crude glycerol conversion to 1,3-PD
was also tested on a 200-L scale. For this purpose,
sterilization of glycerol feedstock was omitted to reduce

20 100 50
18 90 45
16
glycerol; 1,3-propanediol [g/L]

80 40
optical density (605 nm)
14 70 35
12 60 30
10 50 25
8 40 20
6 30 15
4 20 10
2 10 5
0 0 0
0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35
time [h] time [h]

Fig. 2 Acid concentrations during 1-L fed-batch fermentation of Fig. 3 1-L fed-batch fermentation of AKR102a on pretreated crude
AKR102a on pure glycerol: butyric acid (closed triangle, grams per glycerol: 1,3-propanediol concentration (open square, grams per liter),
liter), acetic acid (closed square, grams per liter), and lactic acid (open glycerol concentration (closed circle, grams per liter), and formation
circle, grams per liter) of biomass (closed star, optical density at 605 nm)
Appl Microbiol Biotechnol (2012) 93:10571063 1061

14
The course of acid formation (Fig. 6) almost mirrors the
butyric acid; acetic acid; lactic acid [g/L]

12
1-L fermentation with final concentrations being 11.7 g/L
butyric acid, 5.2 g/L acetic acid, and 5.6 g/L lactic acid.
10

8 Discussion

6
4
2 and 2.3 g/(L*h) (CG), these are among the highest values
0 examples for microbial fed-batch production of 1,3-PD
0 5 10
Fig. 4 Acid concentrations during 1-L fed-batch fermentation of
AKR102a on pretreated crude glycerol: butyric acid (closed triangle,
grams per liter), acetic acid (closed square, grams per liter), and lactic
acid (open circle, grams per liter)
the experimental effort and examine the feasibility of this
method. Additionally, technical requirements limited the
base concentration to 10%, and there was no online
monitoring system available. After starting with a glycerol
concentration of 20 g/L (Fig. 5), feed addition was
repeatedly manually reset and adapted to the ongoing
process. Maximum productivity of 5.0 g/(L*h) was reached
between 6 and 9 h after the start of fermentation. The final
1,3-PD concentration was 61.5 g/L at 30 h with a residual
glycerol concentration of 10.5 g/L. Subsequently, the
overall productivity amounted to 2.1 g/(L*h). Production
yields of 0.530 g1,3-PD/gGly or 0.641 mol1,3-PD/molGly are
similar to the values from the previous small-scale
fermentations.
During this study, 1,3-PD concentrations of 93.7 g/L (PG)
and 76.2 g/L (CG) could be obtained. Together with the
corresponding overall productivities of 3.3 g/(L*h) (PG)
reported for natural producers so far. A selection of
15 20 25 30 35
using different types of carbon sources is given in Table 1.
time [h]
The productivity during early stages of the fermentation
does not seem to be affected by the type of glycerol. A
difference of the growth-associated 1,3-PD production can
be observed after surpassing a concentration of approxi-
mately 60 g/L. It can be assumed that this and the lower
final 1,3-PD concentration can be primarily attributed to
impurities present in the crude glycerol such as fatty acids,
heavy metal ions, or salts. Unsaturated free fatty acids have
been reported to have a negative effect on cell viability
(Rehman et al. 2008; Chatzifragkou et al. 2010). After the
removal of the floating layer, residues of fatty acids probably
still remain within the crude glycerol. Residual heavy metal
ions from biodiesel production might also negatively
influence cell viability (Gonzlez-Pajuelo et al. 2004).
Furthermore, high sodium ion concentrations are said to
inhibit cell growth (Colin et al. 2001). This issue has been
addressed during this study by changing the base from
sodium hydroxide to ammonium hydroxide. At this point
further, experiments have to be carried out to determine the
14
butyric acid; acetic acid; lactic acid [g/L]

70 35
12
60 30
glycerol; 1,3-propanediol [g/L]

10
optical density (600 nm)

50 25
8
40 20
6
30 15

20 10 4

10 5 2

0 0 0
0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35
time [h] time [h]

Fig. 5 200-L fed-batch fermentation of AKR102a on crude glycerol Fig. 6 Acid concentrations during 200-L fed-batch fermentation of
(not sterilized): 1,3-propanediol concentration (open square, grams per AKR102a on crude glycerol: butyric acid (closed triangle, grams per
liter), glycerol concentration (closed circle, grams per liter), and liter), acetic acid (closed square, grams per liter), and lactic acid (open
formation of biomass (closed star, optical density at 605 nm) circle, grams per liter)
1062 Appl Microbiol Biotechnol (2012) 93:10571063

Table 1 Summary of high-level fed-batch production of 1,3-propanediol (1,3-PD) from literature, yield (Y1,3-PD/Gly ) and overall productivity
(Q1,3-PD ) using different microorganisms and carbon sources

Species Strain Carbon 1,3-PD Y1,3-PD/Gly Q1,3-PD Reference

source (g/L) (g/g) (g/(L*h)

Clostridium butyricum DSM 5431 PG 42.0 0.51 2.14 Rehman et al. (2008)
CG 45.0 0.49 2.00
Clostridium butyricum DSM 5431 PG 58.0 0.56 2.70 Gnzel et al. (1991)
Clostridium butyricum E5 PG 65.6 0.54 1.37 Petitdemange et al. (1995)
CG 58.4 0.54 1.22
Clostridium butyricum VPI 1718 CG 67.9 0.55 0.78 Chatzifragkou et al. (2011)
Clostridium butyricum 86.6 1.88 Hartlep and Zeng (2002)
Clostridium diolis DSM15410 PG 78.5 0.53 2.82 Otte et al. (2009)
GSHM 2a CG 53.7 0.40 1.92
Clostridium species IK124 PG 87.7 0.54 1.90 Hirschmann et al. (2005)
CG 80.1 0.56 1.80
Clostridium species IK123 PG 103.2 0.59 1.90 Bock (2004)
Escherichia coli K12 ER2925b PGc 104.4 0.90 2.61 Tang et al. (2009)
Escherichia coli K12b Glucose 135.0 0.51 3.50 Nakamura and Whited (2003)
Klebsiella pneumoniae M5a1 CG 58.8 0.44 0.92 Cheng et al. (2007)
Klebsiella pneumoniae DSM 2026 PG 61.9 0.40 2.00 Mu et al. (2006)
CG 53.0 0.39 1.70
Klebsiella pneumoniae DSM 4799 CG 80.2 0.45 1.16 Jun et al. (2010)
Klebsiella pneumoniae HR526 PGd 95.4 0.40 2.00 Xu et al. (2009)
LDH526b d 102.1 0.43 2.10
Clostridium butyricum AKR102a PG 93.7 0.52 3.30 This study
CG 76.2 0.51 2.30
a
Mutant
b
Genetically modified
c
Two-stage process
d
Aerobic

cause of the positive effect the hydrotalcite treatment seems
to have on the crude glycerol utilization.
A certain dilution effect due to the high water content of
the used crude glycerol in comparison to the pure glycerol
fermentation cannot be ruled out. This is, however, deemed
acceptable since it was the least purified and therefore
cheapest glycerol available.
It was observed that the acid formation shifted from butyric
acid towards lactic acid during fermentations with crude
glycerol. The reasons for this change in acid production can
only be speculated on, since different individual factors or a
combination of those could have contributed to this effect.
The phases of very low glycerol supply resulting from process
operation might have caused a shift in metabolism. In
addition, contents of the used crude glycerol could have
somehow hampered the butyric acid formation from acetyl-
CoA since acetate formation does not seem to be affected.
This could possibly be explained by the accumulation of
impurities or an as yet unspecified limitation caused by media
dilution due to the high water input resulting from the low
glycerol concentration of the crude glycerol. For Clostridium
pasteurianum, for instance, it has been shown that an iron
limitation led to an increase in lactate production when
grown on glycerol (Dabrock et al. 1992). During this study,
the aggregated production of acids in correlation to 1,3-PD
remained similar when compared to the fermentation using
pure glycerol ranging from 0.3 to 0.35 molacids/mol1,3-PD.
When looking at the proposed metabolism (Zeng 1996) and
taking into account the produced acids, the amount of NADH2
regenerated through formation of 1,3-PD compared to those
by butyric and lactic acid remains roughly stoichiometric.
This indicates that the recovery of reduction equivalents
seems to have shifted to the formation of lactic acid since the
pathway to butyric acid is somehow altered or affected.
The obtained 1,3-propanediol concentration of the 200-L
scale fermentation is lower compared to the small-scale
fermentation using crude glycerol albeit yields were slightly
higher. It has to be noted that based on the final volume of
the fermentation broth, approximately 3.5 g/L yeast extract
was used during this experiment whereas 10.5 g/L was used
for the 1-L scale fermentation. Furthermore, the diluted
base (10% versus 25%) raised the amount of water input to
the fermenter. With continuative optimization of process
operation, e.g., implementation of automated feed, it would
be possible to further increase final product concentration
and productivity of the large-scale fermentation.
Appl Microbiol Biotechnol (2012) 93:10571063
Despite application of a simplified glycerol pretreatment,
the use of non-sterile crude glycerol as feed, and a
significant reduction of yeast extract, it was possible to
successfully scale up the fermentation to 200-L scale while
still attaining a sizable amount of 61.5 g/L 1,3-PD.
Together with the use of low-priced crude glycerol, the
applied changes will help to further reduce the overall
process costs due to lower amounts of raw materials used
and to minimize energy costs for sterilization of feedstock.
Acknowledgments This project was funded by the German Federal
Ministry of Education and Research (BMBF) (IG-Biotech 0315026E). I
want to thank PD Dr. Udo Rau and Wolfgang Gral from the Technische
Universitt Braunschweig for the kind technical provision of the large-
scale fermenter and the assistance during the 200-L fermentation.
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