Chinese J. Chem. Eng., 14(1) 132—136 (2006)
RESEARCH NOTES
Production of 2,3-Butanediol by Klebsiella Pneumoniae Using Glucose
and Ammonium Phosphate
QIN Jiayang(秦加阳), XIAO Zijun(肖梓军), MA Cuiqing(马翠卿), XIE Nengzhong(谢能中),
LIU Peihai(刘培海) and XU Ping(许平)*
State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, China
Abstract The production of 2,3-butanediol by Klebsiella pneumoniae from glucose supplemented with different salts
was studied. A suitable medium composition was defined by response surface experiments. In a medium containing glucose and (NH4)2HPO4, the strain could convert 137.0g of glucose into 52.4g of 2,3-butanediol and 8.4g of acetoin in
shaking flasks. The diol yield amounted to 90% of its theoretical value and the productivity was 1—1.5g·L－1·h－1. In
fed-batch fermentation, the yield and productivity of diol were further enhanced by maintaining the pH at 6.0. Up to
92.4g of 2,3-butanediol and 13.1g of acetoin per liter were obtained from 215.0g of glucose per liter. The diol yield
reached 98% of its theoretical value and the productivity was up to 2.1g·L－1·h－1.
Keywords 2,3-butanediol production, optimization, fed-batch, Klebsiella pneumoniae
1
INTRODUCTION
Interest in microbial production of 2,3-butanediol
has been increasing recently due to the extensive industrial application of this product. This colorless and
odorless liquid with a high boiling point and a low
freezing point is a potential valuable fuel additive. Its
－
heating value is 27.198kJ·g 1, which is quite near the
－
value of ethanol (29.055kJ·g 1). Besides, condensation
of diol to methyl ethyl ketone (MEK) coupled with
subsequent hydrogenation yields octane isomers that
can be used to produce high-grade aviation fuel. Currently, the manufacturing of 2,3-butanediol is still
growing by an annual rate of 4%—7% due to the increased demand for polybutylene terephthalate resin,
γ-butyrolactone, spandex, and their precursors[1―5].
In the production of 2,3-butanediol via a mixed
acid pathway, acetoin is an intermediate prior to the
formation of 2,3-butanediol during fermentation. The
metabolic conversion of acetoin to 2,3-butandiol is
reversible. The net reactions forming 2,3-butanediol
and acetoin from glucose are
Glucose
butanediol＋2CO2＋NADH2＋2ATP
Glucose
acetoin＋2CO2＋2NADH2＋2ATP
Therefore, on the mass basis, the theoretical yield of
－
2,3-butanediol and acetoin from glucose are 0.5g·g 1
－1
and 0.49g·g , respectively.
Many investigations have been carried out on microbial production of 2,3-butanediol in the past few
decades, and high-concentration production of
2,3-butanediol have been reported frequently[6―10].
However, some of them used organic substrates as nitrogen sources, and some used new techniques like membrane bioreactor with cell recycle. The former was costly
and difficult for downstream processes, while the latter
needed some complex equipment, which might be expensive in practical industrial production. The aim of our
work was to establish a feasible method for industrial
production of 2,3-butanediol. A medium containing glucose and ammonium phosphate was formulated to produce 2,3-butanediol by Klebsiella pneumoniae. With
fed-batch fermentation, the yield of 2,3-butanediol plus
－
acetoin reached 105.4g·L 1.
2 MATERIALS AND METHODS
2.1 Microorganism and media
The organism used in this study was Klebsiella
pneumoniae CICC 10011 (bought from the China
Center of Industrial Culture Collection). The strain
was maintained on nutrient agar slants. The slants
were incubated at 37℃ for 14h and the fully grown
slants were stored at 4℃.
The medium for inoculation contained: glucose,
－
－
－
80.0g·L 1; (NH4)2HPO4, 6.0g·L 1; KCl, 1.8g·L 1; EDTA,
－
－
0.51g·L 1; MgSO4·7H2O, 0.6g·L 1; FeSO4·7H2O,
－1
－
0.0225g·L ; ZnSO4·7H2O, 0.0075g·L 1; MnSO4·7H2O,
Received 2005-04-11, accepted 2005-12-13.
* To whom correspondence should be addressed. E-mail: [email protected]. cn
133
Production of 2,3-Butanediol by Klebsiella Pneumoniae Using Glucose and Ammonium Phosphate
－
－
0.0038g·L 1; citric acid, 0.21g·L 1; sodium citrate,
－
0.294g·L 1[11]. A loop of cells was inoculated into 50ml
preculture with the above medium in 500-ml flask and
incubated for the desired time at 37℃ with shaking at
－
200r·min 1 on a reciprocal shaker. The optimum inoculum age was 12h with a dry cell weight (DCW) at about
－
8g·L 1.
The media was altered to determine the optimal
formulation for subsequent experiments, and the best
media for the shaking-flask and fed-batch fermentations
were determined as described later in Section 3.2. All
media were sterilized at 115℃ for 20min.
For shaking flasks fermentations, 5ml inoculum
was transferred into 100ml preculture with the best
medium in each 500-ml flask. Fed-batch fermentation
was performed in a 15-L fermentor with a total work－
ing volume of 10L at an impeller speed of 300r·min 1
－1
and an aeration rate of l.0L·min . Temperature was
controlled at 37℃, and the pH value was maintained
at 6.0 by adding KOH or H3PO4. The fermentor was
filled with the optimized medium without glucose,
and sterilized in situ at 115℃ for 20min. The carbon
source was sterilized separately and then added to the
fermentor. For inoculation, 500ml inoculum was
transferred into the fermentor. At regular time intervals, samples were removed from the fermentor to
determine the cell dry weight, glucose and diol concentrations.
Analytical methods
The optical density (OD) of the culture was assayed using a spectrophotometer (UV-340, Beckman)
at 620nm with appropriate dilution. The value of the
optical density was converted to dry cell weight
(DCW) using a calibration equation (DCW ＝
0.5923×OD＋1.9774).
For quick estimation of the amount of glucose
during fermentation, a lactate analyzer (YSI model
2700, USA) was used with a glucose oxidase enzyme
electrode[12].
Determination of the products was carried out on a
Varian gas chromatograph (GC) 3800. The GC was
equipped with a flame ionization detector and a 30m
capillary column operated with N2 as carrier gas. The
temperatures for the GC were as follows: injector temperature, 280℃; detector, 220℃; initial oven tempera－
ture, 50℃ for 1min, followed by 10℃·min 1 ramp to
180℃ for a final 10min hold. The sample was firstly
extracted by butyl acetate, and then injected into the gas
chromatograph. The concentration of the products was
determined by calibration curves.
The response surface experiments were analyzed
with the software “Statistica for windows, Release
4.5A of StatSoft, Inc”.
3 RESULTS AND DISCUSSION
3.1 Gas chromatogram of 2,3-butanediol and acetoin
As shown in Fig.1, two isomers of 2,3-butanediol
could be separated obviously (Though there are three
stereoisomers of 2,3-butanediol, its dextro-form was
not found during fermentation). The main product of
glucose fermentation by Klebsiella pneumoniae was
m-2,3-butanediol. This was quite different from another 2,3-butanediol production strain, Bacillus polymxa, which mainly produced D-2,3-butanediol[13].
The accumulation of L-2,3-butanediol during our fermentation experiments may be that it was formed
from L-acetoin by reduction of diacetyl, which was
catalyzed by NADPH-dependent diacetyl reductase, as
proposed by Ui et al.[14].
2.2
Figure 1
Gas chromatogram of 2,3-butanediol
fermentation
3.2
Culture optimization
For response surface experiments[15], Table 1 was
constructed to determine the optimal concentration of
(NH4)2HPO4 and MgSO4·7H2O, which were the most
important factors in the fermentation. Glucose was
－
increased to 135.0g·L 1; the other ingradients
remained at the same levels as those in section 2.1.
Results were shown in Fig.2.
The analysis suggests the following response
surface:
2
Z = −94.514 + 100.37 x + 104.938 y − 0.202 x −
2
0.49 xy − 53.175 y
from which the optimal concentration of (NH4)2HPO4
－
and MgSO4·7H2O was determined as 24.0g·L 1 and
Chinese J. Ch. E. 14(1) 132 (2006)
Chinese J. Ch. E. (Vol. 14, No.1)
134
－
0.88g·L 1, respectively.
Table 1
The response surface experiment design
x, g·L－1
y, g·L－1
z, g·L－1
(NH4)2HPO4 MgSO4·7H2O production
Block
1
2
1
1
－1
－1
10
0.2
6.1
2
1
1
－1
30
0.2
44.4
3
1
－1
1
10
1.2
27.2
4
1
1
1
30
1.2
55.7
5
1
0
0
20
0.7
62.2
6
1
0
0
20
0.7
62.1
7
2
－1.41
0
5.86
0.7
2.7
8
2
1.41
0
34
0.7
46.4
9
2
0
－1.41
20
0
21.0
10
2
0
1.41
20
1.4
56.4
11
2
0
0
20
0.7
72.1
12
2
0
0
20
0.7
67.0
Figure 3 Graph of growth (DCW), residual glucose (RG)
and diol production during fermentation in shaking flasks
● DCW, g·L－1; ▲ RG, g·L－1
concentration, g·L－1: ■ acetoin; ▼ 2,3-butanediol;
★ 2,3-butanediol+acetoin
In the course of 2,3-butanediol fermentation in
shaking flasks, 135.0g of glucose in total 10 flasks
was transformed into 52.4g of 2,3-butanediol and 8.4g
of acetoin by Klebsiella pneumoniae. The diol yield
reached 90% of its theoretical value and the produc－
－
tivity was 1—1.5g·L 1·h 1.
3.4
Figure 2 Effects of (NH4)2HPO4 and MgSO4·7H2O on the
diol production (Z =－94.514+10.037x+104.938y－0.202xx－
0.49xy－53.175yy)
In summary, the optimized medium contain: glucose,
－
－
135.0g·L 1; (NH4)2HPO4, 24g·L 1; MgSO4·7H2O,
－1
－1
－
0.88g·L ; KCl, 1.8g·L ; EDTA, 0.51g·L 1; FeSO4·7H2O,
－
－
0.0225g·L 1; ZnSO4·7H2O, 0.0075g·L 1; MnSO4·7H2O,
－1
－1
0.0038g·L ; citric acid, 0.21g·L ; sodium citrate,
－
0.294g·L 1. The medium was used for the latter experiments.
Shaking flasks fermentation
As shown in Fig.3, the production of 2,3-butanediol
was a growth-associated phenomenon. The condition for
the maximum product formation was approximately the
same as that for maximum biomass yield. This was the
same as previously reported[16]. In about 50h, all the
glucose was utilized. About 5h after that, 2,3-butanediol
reached its maximum yield.
Fed-batch fermentation
In fed-batch fermentation, the initial glucose con－
centration was 135.0g·L 1. During the fermentation,
glucose was added to the batch at the time when the
consumption of glucose was the most efficient and the
－
residual glucose concentration was below 30.0g·L 1.
Each time 500.0g of solid glucose was added and in
total about 1000.0g of glucose was added.
In about 50h, up to 92.4g of 2,3-butanediol and
13.1g of acetoin per liter were obtained from 235.0g
of glucose per liter with the result shown in Fig.4. The
diol yield reached 98% of its theoretical value and the
－
－
productivity was 2.1g·L 1·h 1.
3.3
February, 2006
Figure 4 Graph of growth (DCW), residual glucose
(RG) and diol production during fed-batch fermentation
● DCW; ▲ RG
concentration, g·L－1: ■ acetoin; ▼ 2,3-butanediol;
★ acetoin+butanediol
Production of 2,3-Butanediol by Klebsiella Pneumoniae Using Glucose and Ammonium Phosphate
Table 2
135
Comparison of cultivation systems for diol production
2,3-Butanediol＋Acetoin
Organism
Medium
Method
yield
－1
g·L
A. aerogenes
K. pneumoniae
A. aerogenes
K. pneumoniae
K. oxytoca
P. polymyxa
K. pneumoniae
K. pneumoniae
glucose and urea
glucose and yeast
extract
glucose and
(NH4)2SO4
glucose and
(NH4)2SO4
glucose and
molasses
glucose, yeast extract and tryptone
glucose and
(NH4)2SO4
glucose and
(NH4)2HPO4
g·L ·h
－1
－1
g·g
fed-batch
111.0
0.90
0.36
[6]
double fed-batch
113.0
0.94
0.50
[7]
cell recycle
110.0
5.4
0.48
[8]
cell recycle
39.6
9.84
0.43
[20]
fed-batch
102.9
1.1
0.49
[9]
fed-batch
57.3
0.96
0.41
[10]
35.0
3.50
25.0
4.25
0.42
[21]
105.5
2.1
0.49
Our research
continuous
fed-batch
At the end of the fermentation, the residual glu－
cose was at 18g·L 1, maybe because the nitrogen resource or other nutrition components were not enough
for keeping the activity of the biomass.
3.5
Ref.
productivity
－1
Discussion
It is known that three enzymes are involved in
the 2,3-butanediol pathway[17,18]: α-acetolactate synthase, α-acetolactate decarboxylase, and acetoin (diacetyl) reductase (also called butanediol dehydrogenase). To study the mechanism of these enzymes, an
inorganic medium is necessary. Pousen and Stougaard
found that the acetolactate synthase was dependent of
Mg2+[19]. This was consistent with the important role it
played in our medium. (NH4)2HPO4 was used as the
main nitrogen source in our medium, but its effect was
not only that. (NH4)2SO4 had ever been used to replace it, but the DCW and the diol yield in that fermentation test were quite low. HPO42- might play an
important but unknown role. More research in this
respect seems necessary.
In the present work, from 215.0g of glucose
about 92.4g of 2,3-butanediol and 13.1g of acetoin per
liter were obtained. The diol yield reached 98% of its
－
－
theoretical value and the productivity was 2.1g·L 1·h 1.
Compared with other reports shown in Table 2, the
yields are on the same level, but the productivity of
our process is much higher except for that reported by
Zeng et al.[8], who used a membrane bioreactor with
cell recycle. In comparison with their process, ours is
much simpler and less costly. Besides, because the
present medium is inorganic, the recovery of the
product from the fermentation broth will be more
economical.
In conclusion, though the yield and the productivity are not the highest, our process is relatively
simpler and highly efficient, possibly feasible for industrial production of 2,3-butanediol.
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