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Chiang Mai J. Sci. 2016; 43(4)
Chiang Mai J. Sci. 2016; 43(4) : 718-725
http://epg.science.cmu.ac.th/ejournal/
Contributed Paper
1, 3-Propanediol Production from Crude Glycerol by
Klebsiella pneumoniae
Jiaxin Wu*[a], Fenghuan Wang[b], Zheng Wang[b], Hua Ye[c] and Peiyi Liu[d]
[a] Key Laboratory of Biological Products and Chemical Drugs for Animals, Ministry of Agriculture,
Beijing Engineering Research Center of Design and Development of Synthetic Peptide Vaccines for
Animals, China Animal Husbandry Industry Co.,Ltd, Beijing 100095, China.
[b] Beijing Key Lab of Bioprocess, College of Life Science and Technology, Beijing University of Chemical
Technology, Beijing 100029, China.
[c] Novozymes (China) Investment Co., Ltd, Beijing 100085, China.
[d] Beijing Gulong Jialan Biotechnology Co.,Ltd, Beijing 100097, China.
*Author for correspondence; e-mail: [email protected]
Received: 18 December 2014
Accepted: 30 April 2015
ABSTRACT
Crude glycerol is a major byproduct of biodiesel manufacture, and can be fractionally
distilled. We present here the microbial conversion of crude glycerol into 1, 3-propanediol by
Klebsiella pneumoniae under micro-aerobic conditions. We evaluated the effects of pure and
crude glycerol on microbial growth and resulting product through batch culture fermentation.
In addition, we studied effect of fatty acid and methyl ester on fermentation. Comparison of
final concentrations and 1, 3-propanediol molar yields indicate that the crude glycerol from
biodiesel production can be used as cheap raw material in the production of 1, 3-propanediol
by Klebsiella pneumoniae. Using pure glycerol, concentration reaches 81.10 g/L and yield is
0.62 mol/mol, while crude glycerol from biodiesel production yields 76.85g/L concentration
and 0.57mol/mol yield. Fatty acid shows itself favorable for 1, 3-propanediol production,
while methyl ester causes strong inhibition of 1,3-propanediol production.
Keywords: 1, 3-propanediol, Klebsiella pneumoniae, biodiesel, methyl ester, fatty acid
1. INTRODUCTION
Biodiesel has become more attractive
recently because of its environmental
benefits and the fact that it is made from
renewable resources. The most common way
to produce biodiesel is by chemical or
enzymatic transesterification. Chemical
methods rapidly yield a high conversion
rate of triacylglycerols to methyl esters[1].
However chemical transesterification recovers
glycerol poorly and produces catalystalkalinized waste water in large volumes.
By aid of various of immobilized lipases,
enzymatic transesterification is also performed
in solvent or in solvent-free media[2-6].
The stoichiometry of methanolysis reaction
requires 3 mol of methanol and 1 mol of
triglyceride to yield 3 mol of fatty acid
methyl ester and 1 mol of glycerol[7], the main
Chiang Mai J. Sci. 2016; 43(4)
by-product of biodiesel industries. Efficient
production requires this by-product be
recaptured and used.[8,9]
1, 3-Propanediol as a product can be
used for synthesis reactions, in particular as a
monomer for polycondensation to produce
polyesters, polyethers and polyurethanes
[10]. Traditional chemical conversion of
acrolein into 1, 3-propanediol requires high
temperature, high pressure and expensive
catalysts. Therefore, much attention has
recently been paid to the microbial conversion
of glycerol to 1, 3-propanediol a relatively
simple process without toxic by-products.
Glycerol can be converted to 1, 3-propanediol
by many microorganisms such as Klebsiella
pneumonia, Bacillus welchi, Lactobacillus ssp.,
Enterobacter spp., Citrobacter spp., and Clostridia
spp.[11] Klebsiella pneumoniae is a typical
microbial strain which is capable of producing
1, 3-propanediol from glycerol.[12] With
glycerol produced from jatropha biodiesel
process, the final concentration of 1,
3-propanediol reaches 56 g/L.[13] Using
fed-batch fermentation by Klebsiella
pneumoniae AK/pConT, the maximum
level of 1, 3-propanediol production from
crude glycerol are 25.9g/L.[14] Production
of ethanol and d-lactate from crude glycerol
by Klebsiella pneumoniae is investigated,
too.[15,16] 1, 3-Propanediol (1,3-PD) is
produced by Klebsiella pneumoniae using
crude glycerol obtained from biodiesel
production without any prior purification.
The 1, 3-Propanediol concentration of
51.3 g/L on crude glycerol from alkalicatalyzed methanolysis of soybean oil
is comparable to that of 53 g/L on crude
glycerol derived from a lipase-catalyzed
process.[17] The production of 1, 3propanediol from industrial glycerol
sources has already been studied, but the
final concentration remains unsatisfied.
Enhancement of 1,3-propanediol production
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by expression of pyruvate decarboxylase
and aldehyde dehydrogenase from Zymomonas
mobilis in the acetolactate-synthase-deficient
mutant of Klebsiella pneumoniae is studies. [18]
Klebsiella pneumoniae (LDH526) lacking
fermentative d-lactate dehydrogenase(LDH)
ferments glycerol to 1,3-propanediol in a
higher 1,3-propanediol concentration[19].
These methods may prove to be a promising
alternative for enhancing the industrial
production of 1,3-propanediol from crude
glycerol.
In this investigation, We intended to
produce 1,3-Propanediol from industrial
biodiesel crude glycerol through microbial
fermentation processes. We employed only
distillation for the production of crude
glycerol from the biodiesel production.
As well, we observed the effect of fatty acid
and methyl ester on the fermentation was also
studied.
2. MATERIALS AND METHODS
2.1 Bacterial Strains
We employed the bacterium, Klebsiella
pneumoniae(0701Y), which provided by
Beijing Key Lab of Bioprocess, Beijing
University of Chemical Technology.
2.2 Culture Conditions
Biodiesel-produced glycerol was
provided by a pilot-scale biodiesel plant
(Beijing University of Chemical Technology).
The source of Biodiesel-produced glycerol
was from soybean oil biodiesel process.
After sterilizing the medium at 116 °C
for 25min, we cultured our strain in a flask
medium consisting of: 3 g/L Yeast extract,
5 mg/L FeCl 2 , 8 g/L glucose, 0.5 g/L
MgSO 4 ⋅7H 2 O, 3.4 g/L K 2 HPO 4 ⋅3H 2 O,
1.3 g/L KH2PO4, 0.5 g/L CaCO3, 4 g/L
(NH4)2SO4, 40 g/L pure or crude glycerol,
1 ml/L trace elements solution. Our
fermentation medium recipe contained
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3 g/L Yeast extract, 5 mg/L FeCl2, 8 g/L
glucose, 0.5 g/L MgSO 4⋅7H 2O, 3.4 g/L
K2HPO4⋅3H2O, 1.3 g/L KH2PO4, 0.5 g/L
CaCO3, 4 g/L (NH4)2SO4, 20 g/L pure or
crude glycerol, and 1 ml/L trace elements
solution. The medium was sterilized at
116 °C for 25min. The solution of trace
elements we used consisted of 70 mg/L
ZnCl2,0.1 g/L MnCl2⋅4H2O, 60 mg/L H3BO3,
0.2 g/L CoCl2⋅2H2O, 20 mg/L CuCl2⋅2H2O,
25 mg/L NiCl 2 ⋅6H 2 O, 35 mg/L
Na2MoO4⋅2H2O, and 0.9 ml HCl (37%). For
feed medium, we used 600 g/L pure or crude
glycerol and 75 g/L glucose after sterilizing
the medium at 116 °C for 25min.
2.2.1 Flask fermentation
We grew cultures on plates at 37 for 24h
before flasking. We then inoculated shake
flasks (250 mL) containing 50 mL of
activation medium with single colonies
and cultured these at 37 °C for 36 hr at
170 rpm in a rotary shaker.
2.2.2 Batch fermentation
We began batches by inoculating shake
flasks (250 mL) containing 50 mL of
activation medium with single colonies
and cultured them at 37 °C for 16 hr at
170 rpm in a rotary shaker. The fermentation
tank had a working volume of 30L
(B. Braun, Germany). We added 15L of
fermentation medium to the tank and
inoculated this with 200mL K. pneumoniae
from shake flask cultures. Temperature was
controlled automatically at 37 °C, and
rotation speed was 180 rpm. With 5 M KOH,
we adjusted pH to 7.0. We bubbled 0.4 vvm
of air into the broth to maintain aerobic
conditions. The dissolved oxygen level was
not more than 15%.
2.2.3 Calculation of yield
Molar yield=(1, 3-Propanediol’s molar
Chiang Mai J. Sci. 2016; 43(4)
mass)/(Glycerol’s molar mass)
2.3 Analytical Methods
2.3.1 Determination of the crude glycerol
by gas chromatography(GC) [20]
On a gas chromatograph GC, Shimadzu,
Japan), fitted with a flame ionizing detector
and capillary column (DB-1ht from J&W
Scientific, 30m × 0.25 mm, 0.2 μ m film
thickness), we analyzed triglyceride, diglyceride,
monoglyceride, free fatty acid and fatty
acid methyl ester content of our samples.
We performed injection in split mode (1/5),
with the injector temperature at 350 °C and
the detector at 360 °C. Oven temperature
was 100 °C during sample (1 μL) injection.
Next, we heated the oven at 15 °C/min to
180 °C, then at 10 °C/min to 230 °C and at
20 °C/min to 330 °C (holding for 5 min),
using nitrogen as the carrier gas at
6.21 mL/min flow rate.
2.3.2 Determination of metabolites by
high-performance liquid
chromatography(HPLC) [19]
Applying HPLC(Shimadzu, Japan), we
analyzed the metabolites in fermentation
broths with a refractive index detector, and a
Bio-Rad Amines HPX-87H organic acids
column, while holding column temperature
at 65 °C, flow rate at 1 ml min-1 and mobile
phase at 5mM H2SO4. Prior to analysis we
filtered samples through 0.45 μm membranes.
3. RESULTS AND DICUSSION
3.1 Crude Glycerol from the Biodiesel
Production
We synthesized fatty acid methyl esters
from direct transesterification of waste
edible oils by Candida antarctica lipase, where
the corresponding triglycerides react with
methanol in solvent and in solvent-free
media. The process yields glycerol by-product,
which we separated using a hydrocyclone
Chiang Mai J. Sci. 2016; 43(4)
[20]. However, the concentration of glycerol
is too low in eluate from biodiesel
production, and proved complicated to
work with, particularly the toxic effects
of methanol on cell growth, making glycerol
eluate inappropriate for microbiological
transformation to 1,3-propanediol.Therefore,
eluate has to be distilled by scraped-film
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evaporator to concentrate glycerol.
After elimination of methanol, the distilled
eluate is termed ‘crude glycerol’, whose
components are itemized in Table 1.
Only traces of emulsified ester remained
in the crude glycerol.
Table 1. The main components of crude glycerol.
Triglycerides(g/L)
Diglycerides(g/L)
Monoglycerides(g/L)
Free fatty acids(g/L)
Fatty acid methyl ester(g/L)
Glycerol(g/L)
3.2 Shake Flask Cultivation
To investigate whether crude glycerol
from biodiesel industry is appropriate to
support cell growth and how impurities of
the biodiesel industries could affect the
1, 3-propanediol production, we used
commercial-grade glycerol and crude
glycerol at a concentration of 40 g/L for a
carbon source. As shown in Table 2,
2.13
1.60
3.71
4.61
23.96
506.55
we obtained higher 1,3-propanediol
concentration and subsequently equivalent
molar yield with crude glycerol,
demonstrating that crude glycerol is an
adequate carbon source for the growth of
Klebsiella pneumonia and 1, 3-propanediol
production. While fatty additives influences
oxygen transfer.
Table 2. Shake flask fermentation of the pure and crude glycerol by Klebsiella pneumoniae.
Crude glycerol
Pure glycerol
1,3-Propanediol(g/L)
Average
18.65
18.70±0.06
18.70
18.76
Average
16.15
16.7±0.48
16.99
16.96
Molar yield(mol/mol)
Average
0.62
0.63±0.01
0.63
0.63
Average
0.66
0.64±0.02
0.63
0.64
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3.3 Fed-batch Cultures
We further obser ved fed-batch
fermentation and the influence of two
different glycerols on the production of
1,3-propanediol. Again, we conducted the
conversion experiment with pure glycerol
and crude glycerol from the biodiesel
industries as different medium feeds.
Under microaerobic conditions, Klebsiella
pneumoniae can be successfully cultivated and
efficiently converts glycerol to various
products. Part of the glycerol converts to
1,3-propanediol, the remainder is transformed
via dihydroxyacetone to glyceraldehyde-3phosphate and further to pyruvate. The
products of glycerol oxidation arise from
pyruvate cleavage to acetyl CoA and formate,
followed by the formation of by-products,
e.g. ethanol and acetate. The reduction
equivalent (NADH2) generated along
this route may be transferred to an
external electron acceptor, or to the
dehydration products of glycerol, namely
3-hydroxypropionaldehyde[21]. Under
anaerobic conditions, acetate caused the
strong inhibition, while 1,3-propanediol is
also an inhibitory end product. In contrast,
cells grown under aerobic conditions are
more resistant to acetate[22]. Moreover,
glycerol as a carbon source also results
in inhibition. Therefore were chose 1,
3-propanediol, acetate and glycerol as the
critical parameters for further investigation.
The 1, 3-propanediol fed-batch
fermentation with pure glycerol proceeded
under aerobic conditions as shown in
Figure 1. Cells grew exponentially to
maximum concentration, where they rapidly
produced 1, 3-propanediol. While, 1, 3propanediol is associated with cell growth
according to our result, acetate shows
no positive associated with cell growth.
In stationary and decline phase, by-products
accumulate to 2-4 g/L acetate, and
Chiang Mai J. Sci. 2016; 43(4)
approximately 65 g/L 1, 3-propanediol, at
the point when cell growth ceases. From
cessation of cell growth, 1, 3-propanediol
formation slowly increases to maximum
(81.10 g/L 1, 3-propanediol). Molar yield is
0.62 mol/mol.
Figure 1. Fed-batch fermentation of pure
glycerol by Klebsiella pneumoniae.
Our experiments on crude glycerol
from biodiesel production were intended
to discover how higher final concentration
of 1,3-propanediol could be obtained
(Figure 2). The result trends for cell growth,
1, 3-propanediol and acetate by crude
glycerol are similar to those for pure
glycerol. Acetate concentration by crude
glycerol, however, appears to be higher,
whereas the 1, 3-propanediol concentration
is lower than that by pure glycerol.
We achieved a maximum concentration
of 1, 3-propanediol 76.85g/L by crude
glycerol. Molar yield is 0.57 mol/mol.
Compared with crude glycerol without any
prior purification[17], the concentration of
1, 3-propanediol with distilled crude
glycerol is higher. In contrast to shake
flask experiment, the concentration of
1, 3-propanediol in fed-batch fermentation
by using crude glycerol is lower than
pure glycerol, the possible reason is that,
in the process of adding crude glycerol
continuously, the impurities in crude glycerol
Chiang Mai J. Sci. 2016; 43(4)
are continuously cumulated, and the damage
to Klebsiella pneumoniae strain is increased
simultaneously, which finally affects the
concentration of 1,3-propanediol. Meanwhile,
We detected traces of ester (especially fatty
acid methyl ester) and free fatty acids in crude
glycerol after emulsification. the production
of 1,3-propanediol may be affected by fatty
acid methyl ester and/or free fatty acids.
We executed subsequent experiments to study
the effect of methyl ester and free fatty acids
on the production of 1,3-propanediol.
Figure 2. Fed-batch fermentation of crude
glycerol from the biodiesel industries by
Klebsiella pneumoniae.
3.4 Effect of Methyl Ester and Fatty
Acid(oleic acid)on the Fermentation
We conducted fermentations in 40 g/L
pure glycerol with Klebsiella pneumoniae
at various methyl ester and oleic acid
concentrations ranging from 0 to 20g/L.
The 1, 3-propanediol productions are
depicted in Figures 3 and 4. After adding some
oleic acid into the culture medium, we can
see that the production of 1, 3-propanediol
generally increased with the input of oleic
acid, although there is a certain fluctuation
within the increasing process, therefore,
under the little oxygen condition, the oleic
acid can promote the production of 1,
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3-propanediol in some degree. While adding
methyl ester into the culture medium, there
is no obvious change occurred to the
production of 1, 3-propanediol, however it
can be seen that 1, 3-propanediol production
after adding methyl ester are always lower
than the production of 1, 3-propanediol
before adding methyl ester. Therefore, methyl
ester cause inhibition of 1, 3-propanediol
production in some degree. Adding methyl
ester and oleic acid affect oxygen tranfer in
the broth, which influences the concentration
of 1, 3-propanediol through dissolved
oxygen.
Figure 3. Effect of oleic acid on the
fermentation.
Figure 4. Effect of methyl ester on the
fermentation.
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4. CONCLUSIONS
Searching for a commercial-grade
glycerol substitute for conversion to 1, 3propanediol using Klebsiella pneumoniae,
we created this study. One desirable
replacement is the main by-product from
the conversion of waste edible oil into
biodiesel: glycerol. After a simple distillation
step, we applied glycerol eluate from the
biodiesel industry directly to the fermentation
processes. The final concentration and molar
yield of 1,3-propanediol are 81.10g/L
and 0.62mol/mol respectively by using
pure glycerol, whereas concentration is
76.85g/L and molar yield is 0.57mol/mol
using crude glycerol from biodiesel
production. The total product of 1, 3propanediol with crude glycerol is less than
that of pure glycerol, which may be caused
by methyl ester residue in crude glycerol.
In light of biodiesel’s economic and
environmental significance, these fundamental
results will be of significance on the path to
maximal production efficiency. Fatty acid
(oleic, in this case) is favorable for the
production of 1, 3-propanediol, while methyl
ester causes strong inhibition of 1, 3propanediol production.
ACKNOWLEDGEMENTS
The authors thank Professor Tianwei Tan
for guidance and encouragement.
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