Appl Microbiol Biotechnol (2009) 82:49–57
DOI 10.1007/s00253-008-1732-7
BIOTECHNOLOGICAL PRODUCTS AND PROCESS ENGINEERING
Enhanced 2,3-butanediol production by Klebsiella
pneumoniae SDM
Cuiqing Ma & Ailong Wang & Jiayang Qin & Lixiang Li &
Xulu Ai & Tianyi Jiang & Hongzhi Tang & Ping Xu
Received: 21 August 2008 / Revised: 25 September 2008 / Accepted: 25 September 2008 / Published online: 24 October 2008
# Springer-Verlag 2008
Abstract Enhanced 2,3-butanediol (BD) production was
carried out by Klebsiella pneumoniae SDM. The nutritional
requirements for BD production by K. pneumoniae SDM
were optimized statistically in shake flask fermentations.
Corn steep liquor powder and (NH4)2HPO4 were identified
as the most significant factors by the two-level Plackett–
Burman design. Steepest ascent experiments were applied
to approach the optimal region of the two factors and a
central composite design was employed to determine their
optimal levels. The optimal medium was used to perform
fed-batch fermentations with K. pneumoniae SDM. BD
production was then studied in a 5-l bioreactor applying
different fed-batch strategies, including pulse fed batch,
constant feed rate fed batch, constant residual glucose
concentration fed batch, and exponential fed batch. The
maximum BD concentration of 150 g/l at 38 h with a diol
productivity of 4.21 g/l h was obtained by the constant
residual glucose concentration feeding strategy. To the best
Cuiqing Ma and Ailong Wang contributed equally to this work.
C. Ma : A. Wang : J. Qin : L. Li : X. Ai : T. Jiang
State Key Laboratory of Microbial Technology,
Shandong University,
Jinan 250100, People’s Republic of China
H. Tang : P. Xu
Key Laboratory of Microbial Metabolism, Ministry of Education,
School of Life Sciences and Biotechnology,
Shanghai Jiao Tong University,
Shanghai 200240, People’s Republic of China
P. Xu (*)
School of Life Sciences and Biotechnology,
Shanghai Jiao Tong University,
Shanghai 200240, People’s Republic of China
e-mail: pingxu@sjtu.edu.cn
of our knowledge, these results were new records on BD
fermentation.
Keywords Klebsiella pneumoniae . 2,3-Butanediol .
Medium optimization . Fed batch
Introduction
Gradual exhaustion of natural resources has led to the search for
renewable resources for sustainable development and there has
been an increasing interest in 2,3-butanediol (BD) because of
its extensive application in varied fields such as fuel, chemical
industry, food industry, and so on (Xiu and Zeng 2008). BD is
a chiral compound with a high boiling point and a low
freezing point, which is a colorless and odorless liquid at
room temperature (Garg and Jain 1995). As an important
starting material, BD can be used to produce valuable
derivatives such as methyl ethyl ketone and 1,3-butadiene
(Syu 2001). BD has been shown to have the potential to be
used in the manufacture of printing inks, perfumes, fumigants,
moistening and softening agents, explosives and plasticizers,
and as a carrier for pharmaceuticals (Garg and Jain 1995).
BD is produced via a mixed acid fermentation pathway
and differs qualitatively and quantitatively depending on
the strains and species of bacteria and their culture
conditions. Acetoin (AC) is the main by-product during
the fermentation. Many bacterial species such as Klebsiella
pneumoniae (Lee and Maddox 1986), Klebsiella oxytoca
(Afschar et al. 1993), Enterobacter cloacae (Saha and
Bothast 1999), Enterobacter aerogenes (Zeng et al. 1990),
and Bacillus polymyxa (de Mas et al. 1988) can secrete BD.
Among all these strains, K. pneumoniae is one of the best
organisms that have shown the potential for industrial BD
production. K. pneumoniae mainly produces meso-BD and
50 Appl Microbiol Biotechnol (2009) 82:49–57

is often used for the production of BD because of its more
complete fermentation, broad substrate spectrum and
cultural adaptability (Garg and Jain 1995). Several strate-
gies have been widely used to enhance BD production,
such as introducing superproductive strains, optimizing
fermentation operating conditions, and building mathemat-
ical models (Garg and Jain 1995; Lee and Maddox 1986;
Zeng et al. 1990; Syu et al. 1993). Afschar et al. (1993)
achieved a BD concentration of 118 g/l using recycled cells
and the diol (AC + BD) productivity amounted to 2.4 g/l h
by K. oxytoca. Yu and Saddler (1983) obtained a diol
concentration of 113 g/l using fed-batch operation with K.
pneumoniae but the diol productivity was relatively low
(0.94 g/l h). Although BD production has been improved,
the concentration and productivity are not high enough for
economical industrial production. Therefore, it is essential
to further improve BD production by selecting high-yield
strains or systematically optimizing the fermentation
condition.
In this study, a bacterium designated as K. pneumoniae
SDM was isolated from orchard soil samples and demon-
strated good potential for BD production. A sequential
statistical experimental design was used to develop a
suitable fermentation medium. Fed-batch fermentations
were conducted using different feeding strategies in a 5-
l bioreactor and remarkable BD yield and productivity were
obtained.
Materials and methods
Bacterial identification
Classic physiological characteristics were tested according
to Bergey’s Manual of Determinative Bacteriology (Holt et
al. 1994). 16S rRNA gene sequence was amplified
according to standard procedures (Sambrook and Russell
2001) and compared to the sequences in the GenBank
database through BLAST sequence analysis.
Inoculum preparation
Statistical experiment design and data analysis
A three-step experimental design based on statistical
methods was used to optimize the medium for BD
production. The Plackett–Burman (PB) design, an efficient
technique for medium-component optimization (Reddy et
al. 2008), was firstly used to pick factors that significantly
influenced BD production, and insignificant ones were
eliminated to obtain a smaller, more manageable set of
factors. It was based on the first-order model:
X
Y ¼ b0 þ bi Xi ð1Þ
where Y is the response; β0 is the model intercept and βi is
the linear coefficient, and Xi is the level of the independent
variable. Each variable is represented at two levels, high and
low, which are denoted by (+) and (−), respectively. Table 1
illustrates the levels of each variable used in the experimental
design. SAS package (version 9.0, SAS Institute, Cary,
USA) was used to direct the effects and statistical analysis of
the variables. The fit of the regression model obtained was
checked using the adjusted coefficient of determination R-
squared. The significance of each variable was determined
using Student’s t test. In our experiments, the variables with
confidence levels above 95% were considered as influencing
BD production significantly.
Following that, the steepest ascent was generated by the
first-order empirical equation obtained by the PB design to
move rapidly towards the neighborhood of the optimum
response. The center point of the PB design was taken as
the origin for the path of steepest ascent.
Response surface methodology (RSM) was used to
optimize the screened variables for enhanced BD produc-
tion based on central composite design (CCD, Kennedy and
Krouse 1999) with five coded levels. For statistical
calculations, the relationships between the coded values
and actual values are described by the following equation:
xi
x0
Xi ¼ i ¼ 1; 2; . . . ; k ð2Þ
Δxi
where Xi is the dimensionless coded value of the indepen-
dent variable xi; xi is the actual value of that independent

K. pneumoniae SDM was maintained on agar slants Table 1 The PB design for screening variables in BD production
containing the following medium: glucose 15 g/l, peptone
Factors (g/l) Variables Low level (−1) High level (+1)
10 g/l, yeast extract 5 g/l, KCl 5 g/l, and agar at pH 7.0. The
slants were incubated at 37°C for 12 h and then stored at Glucose X1 60 80
4°C. CSLP X2 2 6
The seed culture was prepared by inoculating a full loop (NH4)2HPO4 X3 1 3
of cells from freshly prepared slants into 50 ml of the Sodium acetate X4 1 3
following medium: glucose 20 g/l, (NH4)2HPO4 5 g/l, KCl X5 0.4 0.8
MgSO4 0.3 g/l, KCl 1 g/l, pH 7.0. The cultivation was MgSO4 X6 0.1 0.3
FeSO4·7H2O X7 0 0.02
conducted in 300-ml shake flasks for 10 h with agitation
MnSO4·7H2O X8 0 0.01
(200 rpm, reciprocal shaker) at 37°C.
Appl Microbiol Biotechnol (2009) 82:49–57
variable; x0 is the real value of the independent variable xi
at the center point and Δxi is the step change. The role of
each variable, their interactions, and statistical analysis to
obtain predicted yields is explained by applying the
following quadratic equation:
X X X m
Y ¼ b0 þ bi Xi þ bij Xi Xj þ bii Xi2 ð3Þ
where Y is the predicted response; β0 is the offset term; βi is
the linear effect; βii is the squared effect; βij is the
interaction effect, and Xi is the dimensionless coded value
of xi.
For the flask experiments, 5 ml of the seed culture were
inoculated into 100 ml of the basal medium of fermentation
in 500-ml flasks. The flasks were incubated at 37°C on a
reciprocal shaker at 200 rpm. All experiments were
repeated at least three times. Statistical and numerical
analyses were carried out by means of the analysis of
variance (ANOVA). The 3-D response surface and contour
presentations were plotted using the SAS package.
Batch and fed-batch fermentations
Batch and fed-batch fermentations were conducted in a
5-l bioreactor (BIOSTAT B, B. Braun Biotech International
GmbH, Germany) with 3-l initial medium. The seed culture
prepared previously was inoculated (10%, v/v) into the
optimized fermentation medium with initial pH 7.0. The
cultivation was carried out at 37°C, stirring at 500 rpm, and
airflow at 1.5 vvm. When the pH decreased to 6.0, it was
maintained at 6.0 by automatic addition of 4 M H3PO4 or
6 M KOH using a program-controlled peristaltic pump.
Feed rate of substrate was designed according to the modes
of operation used and the glucose consumption rate.
Samples were collected periodically to determine the
biomass, glucose, and diol concentrations.
Batch fermentation was carried out with a glucose
concentration of 70 g/l. It was used to confirm the
suitability of the model equation for predicting maximum
BD production, to study the kinetic parameters for growth
of K. pneumoniae SDM, and to understand its physiology
in the bioreactor in order to optimize the feed strategy.
All fed-batch fermentations were conducted with an
initial glucose concentration of 70 g/l and the feeding
substrate was pumped into the bioreactor using a computer-
coupled peristaltic pump. In pulse fed-batch fermentation,
glucose was fed into the bioreactor at different pulse
feeding times, when the residual glucose concentration
decreased to 15–20 g/l. In constant feed rate fed-batch
fermentation, when the residual glucose concentration
decreased to 15–20 g/l, glucose solution (800 g/l) was
pumped into the bioreactor at a feeding rate of 20 or 30 ml/
51
h. In constant residual glucose concentration fed-batch, the
residual glucose concentration was maintained at 0–10, 20–
30, or 40–50 g/l by feeding glucose solution (800 g/l).
In exponential fed-batch fermentation, the nutrient
feeding rate was determined by Eq. 4 (Nor et al. 2001):
F¼ ðVX Þ0 expðmt Þ ð4Þ
YX =S ðSi
S Þ
where F is the feeding rate; μ is the specific growth rate;
(VX)0 is the biomass at start of feed; t is the culture time;
YX/S is the theoretical cell yield on substrate; Si and S are
substrate concentrations in the feeding solution and in the
reactor, respectively.
Analytical methods
The glucose concentration of the samples was measured
enzymatically by a bioanalyzer (SBA-40C, Shandong
Academy of Sciences, P. R. China) after centrifuging and
diluting to appropriate concentration.
Dry cell mass concentration was estimated by measuring
the optical density of the sample at 620 nm in a
spectrophotometer (Ultrospec 2100 pro UV/Visible Spec-
trophotometer, Amersham Biosciences, Piscataway, USA)
and by its correlation with the dry cell weight (DCW),
obtained gravimetrically.
Diol in the broth was extracted by ethyl acetate with the
addition of isoamyl alcohol as the internal standard and
then quantified using a GC system (Varian 3800, Palo Alto,
USA) equipped with a flame ionization detector and a 30-m
SPB-5 capillary column (0.32-mm inside diameter, 0.25-
μm film thickness; Supelco, Bellefonte, USA). The
operation conditions were as follows: nitrogen was used
as the carrier gas; the injector temperature and the detector
temperature were both 280°C; and the column oven
temperature was maintained at 40°C for 3 min, then raised
to 240°C at a rate of 20°C/min. The software Star 6.0
Chromatography Workstation was used for data acquisition
and evaluation. The concentration of the products was
determined by calibration curves.
Results
Bacterial identification
The bacterium used in this study was isolated from orchard
soil in Shandong Province, P. R. China. The strain with
highest BD production was obtained by ion beam mutation
and deposited at the China Center for Type Culture
Collection (M 208097). Strain SDM was found to be a
nonmotile, gram-negative, and straight rod-shaped bacteria.
It did not grow at 10°C. Voges–Proskauer test was positive,
52 Appl Microbiol Biotechnol (2009) 82:49–57

as well as urease and lysine decarboxylase. However, Table 3 Effects and statistical analysis of variables
oxidase reaction was negative, so were tests for methyl Variable Coefficient Standard error t value P value
red test, liquefaction of gelatin, and indole production. It
was capable of growing on glucose, lactose, sucrose, Intercept 21.0575 0.6658 31.63 <0.0001
xylose, maltose, arabinose, rhamnose, mannitol, and KCN X1 0.3975 0.6658 0.60 0.5925
X2 3.7758 0.6658 5.67 0.0109
and could utilize citrate and malonate. The organism was
X3 4.9708 0.6658 7.47 0.0050
also identified through BLAST analysis of the partial
X4 1.0842 0.6658 1.63 0.2019
sequences of 16 S rRNA gene. It was 99% identical with X5 −0.4642 0.6658 −0.70 0.5359
some sequence of K. pneumoniae according to its 16 S X6 −0.4308 0.6658 −0.65 0.5637
rDNA sequence (1,506 bp). The sequence was deposited in X7 1.0508 0.6658 1.58 0.2126
the GenBank database with accession no. EU872412. X8 0.9108 0.6658 1.37 0.2648
Based on these results, strain SDM was identified as a
R2 =0.9698, R2 (Adj)=0.8891; significant at the 95% level
strain of K. pneumoniae and designated as K. pneumoniae
SDM.
greatest positive impacts on the production of BD were
identified as X2 (CSLP; p=0.0109) and X3 ((NH4)2HPO4;
Medium optimization p=0.0050). X1 (glucose), X4 (sodium acetate), X7 (FeS-
O4·7H2O), and X8 (MnSO4·7H2O) were set at their high
Plackett–Burman experimental design levels according to the positive effects although they were
nonsignificant to BD production. Factors such as X5 (KCl)
PB design for a total of eight variables was used to identify and X6 (MgSO4) had negative effects and were set at their
which variables have significant effects on BD production low levels.
(Table 1). The medium includes phosphate, acetate, Fe2+, To approach the neighborhood of the optimum response,
Mn2+, and Mg2+, which significantly affect BD production the fitted first-order model equation for BD production was
(Garg and Jain 1995; Syu 2001; Qin et al. 2006). The obtained from the PB design experiments:
medium also contained glucose as carbon source and corn Y ¼ 21:0575 þ 0:3975 X1 þ 3:7758 X2 þ 4:9708 X3
steep liquor powder (CSLP) as nitrogen source according to
the pre-experiments. The upper and lower limits of each þ 1:0842 X4
0:4642 X5
0:4308 X6
variable were chosen according to the preliminary investi-
þ 1:0508 X7 þ 0:9108 X8 : ð5Þ
gation of the limits of the variables.
Table 2 represents the PB experimental design for 12 Statistical testing was carried out using Fisher’s test for
trials with two levels of each variable and the ANOVA according to the experimental data.
corresponding BD production. Table 3 shows the effects The F and p values were 12.02 and 0.0327, respectively.
of the variables on the response and the significant levels. The test model was statistically significant at the 96.7%
Based on the statistical analysis, the factors having the level of significance. The quality of fit of the polynomial
model equation was expressed by the coefficient of
Table 2 The PB design variables (in coded levels) with BD determination (R2), which equaled 0.9698, indicating that
production as response 96.98% of the variability in the response could be explained
by the model. The value of the adjusted determination
Run Variable levels BD (g/l)
coefficient (Adj R2 =0.8891) was also very high which
X1 X2 X3 X4 X5 X6 X7 X8 advocates for a high significance of the model. These
results indicated that the response equation provided a
1 +1 −1 +1 −1 −1 −1 +1 +1 23.68 suitable model for the PB design experiment.
2 +1 +1 −1 +1 −1 −1 −1 +1 21.41
3 −1 +1 +1 −1 +1 −1 −1 −1 26.89
The path of steepest ascent
4 +1 −1 +1 +1 −1 +1 −1 −1 23.80
5 +1 +1 −1 +1 +1 −1 +1 −1 22.14
6 +1 +1 +1 −1 +1 +1 −1 +1 27.52 Based on the model equation obtained, Eq. 5, the path of
7 −1 +1 +1 +1 −1 +1 +1 −1 28.67 steepest ascent was employed to move rapidly towards the
8 −1 −1 +1 +1 +1 −1 +1 +1 25.61 neighborhood of the optimum response, i.e., increasing
9 −1 −1 −1 +1 +1 +1 −1 +1 11.22 CSLP concentration and (NH4)2HPO4 concentration in
10 +1 −1 −1 −1 +1 +1 +1 −1 10.18 order to improve the BD yield. From the coefficient of X2
11 −1 +1 −1 −1 −1 +1 +1 +1 22.37
and X3 (3.7758, 4.9708), which was approximately equiv-
12 −1 −1 −1 −1 −1 −1 −1 −1 9.20
alent to (1, 1.32), the (NH4)2HPO4 concentration (X3)
Appl Microbiol Biotechnol (2009) 82:49–57 53

would increase 1.32 design units (1.32 g/l) if the CSLP Table 5 The results of the central composition experiment
concentration (X2) increased one unit (2 g/l). The center Run Coded variable level Real variable level Y (g/l)
point of the PB design was considered as the origin of the
path. The design and responses of the steepest ascent X1 X2 CSLP (NH4)2
experiment are shown in Table 4. The BD yield decreased (g/l) HPO4 (g/l)
sharply after the third step, with the highest production 1 −1 −1 6 3.32 30.3
being 35.11 g/l, from the combination of CSLP (8 g/l) and 2 +1 −1 10 3.32 33.4
(NH4)2HPO4 (4.64 g/l). This combination was used as the 3 −1 +1 6 5.96 32.3
middle point for the second-order experiment, i.e., CCD. 4 +1 +1 10 5.96 33.4
5 −1.414 0 5.17 4.64 31.1
The central composition experiment and response surface 6 +1.414 0 10.83 4.64 32.5
7 0 −1.414 8 2.77 30.7
analysis
8 0 +1.414 8 6.51 32.0
9 0 0 8 4.64 34.9
A response surface design is appropriate when the 10 0 0 8 4.64 34.8
neighborhood of the optimum response is approached (Li 11 0 0 8 4.64 35.5
et al. 2002). So the two significant independent variables 12 0 0 8 4.64 35.4
(CSLP and (NH4)2HPO4) were further explored using 13 0 0 8 4.64 35.1
CCD. The design matrix of the variables together with the
experimental responses is shown in Table 5. By applying
multiple regression analysis to the experimental data, the CCD experiment and that analysis of the response trends
following second-order polynomial equation was found to using the model was reasonable.
explain the BD production: The significant levels of each variable determined by t
test are shown in Table 6. The Student’s t test and p values
Y ¼ 35:1399 þ 0:4975 X1 þ 0:7549 X2
1:4764 X12 were used as a tool to check the significance of each

0:5000 X1 X2
1:7014 X22 ð6Þ coefficient, which also indicated the interaction strength
between each independent variable. The larger the magni-
where Y is the predicted BD production (g/l); X1 and X2 are tude of the t value and smaller the P value, the more
the coded values of CSLP and (NH4)2HPO4, respectively. significant is the corresponding coefficient (Elibol 2004).
On the basis of the experimental values, statistical testing Among the model terms, the linear and quadratic of
was carried out using Fisher’s test for ANOVA. The F and (NH4)2HPO4 (p<0.01) are more significant than the other
p values were 23.90 and 0.0003, respectively. The test factors. These suggest that the concentration of
model was statistically significant at the 99% level of (NH4) 2HPO 4 has a direct relationship with the BD
significance. The goodness of fit of the quadratic regression production in the medium. Since the concentration of
model equation can be checked by the coefficient of (NH4)2HPO4 is also very significant in the quadratic level
determination (R2). The closer the R2 value is to 1.00, the (p=0.0001<0.001), meaning that it can act as limiting
stronger the model is and the better it predicts the response nutrient and a little variation in its concentration will alter
(Kaushik et al. 2006). The results showed the value of the the BD production. The concentration of CSLP is also very
determination coefficient (R2 =0.9447), indicating that significant in the quadratic level (P=0.0002<0.001). The
94.47% of the variability in the response could be explained interaction between CSLP and (NH4)2HPO4 (X1X2) has a
by the model. The value of the adjusted determination nonsignificant influence on BD production.
coefficient (Adj R2 =0.9052) was also very high to advocate The three-dimensional response surface and contour plot
for a high significance of the model. These results indicated are shown in Fig. 1. It is evident that the response surface is
that the response equation provided a suitable model for the convex in nature suggesting that there are well-defined

Table 6 Significance test of regression coefficient

Table 4 Experimental design and results of the steepest ascent
Variable Coefficient Standard error t value P value
Run CSLP (g/l) (NH4)2HPO4 (g/l) BD (g/l)
Intercept 35.1399 0.2548 137.89 <0.0001
1 4 2 29.75 X1 0.4975 0.2015 2.47 0.0429
2 6 3.32 33.66 X2 0.7549 0.2015 3.75 0.0072
3 8 4.64 35.11 X12 −1.4764 0.2161 −6.83 0.0002
4 10 5.96 34.41 X1X2 −0.5000 0.2849 −1.75 0.1227
5 12 7.28 31.78 X22 −1.7014 0.2161 −7.87 0.0001
54 Appl Microbiol Biotechnol (2009) 82:49–57

between these two results justified the validity of the

response model and the existence of an optimum point.

Batch and fed-batch fermentations

Batch fermentations were conducted to test the suitability of

Fig. 1 Response surface figure (a) and corresponding contour (b) of
the mutual effects of CSLP and (NH4)2HPO4 on 2,3-butanediol
production
optimum conditions. The less prominent or negligible
interaction of CSLP and (NH4)2HPO4 is also shown by
the almost circular nature of the contour plot, which
coincides with the significance test of the regression
coefficient. It is obvious from the plot that BD production
has a maximum point in the studied region. When X1 =
0.13427 (CSLP, 8.27 g/l) and X2 =0.20221 ((NH4)2HPO4,
4.91 g/l), the predicted maximum BD production
corresponding to these values is 35.3 g/l.
To sum up, the optimum medium composition for the
production of BD by K. pneumoniae SDM is as follows:
glucose, 80 g/l; CSLP, 8.27 g/l; (NH4)2HPO4, 4.91 g/l;
sodium acetate, 3 g/l; KCl, 0.4 g/l; MgSO4, 0.1 g/l;
FeSO4·7H2O, 0.02 g/l; MnSO4·7H2O, 0.01 g/l.
Verification by batch culture in shake flasks
the model using the optimal medium. The glucose
consumption, biomass growth, and product synthesis were
monitored during the cultivation. Results showed that the
cells grew rapidly after an adaptation period of 2 h and the
glucose was depleted after 9 h. The maximum concentra-
tion of BD at 34.6 g/l also validated the suitability of the
model. The results of different fed-batch fermentation
methods for BD production are shown in Table 7.
The constant glucose concentration fed-batch process had
the highest concentration and productivity compared with
pulse, exponential, or constant rate fed-batch strategies for BD
fermentation. The high diol concentration (160 g/l) and
biomass (32.1 g/l) indicate that this method may provide a
more suitable environment for the cell metabolism and BD
production. We also compared the effects of different residual
glucose concentrations on BD production and found that the
optimum residual glucose concentration for the maximum BD
concentration was 20–30 g/l (data not shown).
The changes of key variables (residual glucose (RG),
pH, DCW, AC, and BD production) during constant
glucose concentration fed-batch fermentation are shown in
Fig. 2. BD attained a maximal accumulation of 150 g/l and
the diol productivity was 4.21 g/l h, which indicated that
the optimum medium components obtained by statistical
experimental design in shake flasks were also applicable to
overproduction of BD in fed-batch mode.
Discussion
In order to make the production of BD economical on
industrial scale, high concentration and productivity and
Table 7 Comparison of different fed-batch fermentation methods on
BD production
Constant Exponential Pulse Constant
feed rate fed batch fed glucose
fed batch concentration
batch fed batch

To validate the adequacy of the model equation for BD (g/l) 123.2 115.7 141.6 150
predicting maximum BD production, the confirmation BD + AC (g/l) 131.4 124 151 160
experiments were performed using the optimal medium. Productivity 2.92 2.48 3.68 4.21
The experiments yielded an average maximum concentra- (g/l h)
Yield (%) 96.7 94.1 95.3 95
tion of 36.7 g/l, which was closely consistent with the
DCW (g/l) 27.9 24.4 30.3 32.1
model predicted value of 35.3 g/l. The good correlation
Appl Microbiol Biotechnol (2009) 82:49–57
Fig. 2 Time course of fed-batch
fermentation by K. pneumoniae
SDM in a 5-l bioreactor using
the optimized medium. Sym-
bols: filled diamonds, DCW;
filled circles, pH; filled squares,
RG; filled upright triangles, AC;
filled inverted triangles, BD;
empty squares, AC + BD
low cost of fermentation are essential (Garg and Jain 1995;
Xiu and Zeng 2008). Several methods have been used to
achieve this goal as mentioned in the introduction. In
addition to introducing a productive strain, designing an
appropriate fermentation medium is also of crucial impor-
tance to improve the efficiency and productivity of the
fermentation process because product concentration, yield,
and cell growth conditions are strongly influenced by
medium composition such as the carbon source, nitrogen
source, inorganic salts, and so on (Garg and Jain 1995;
Swift et al. 2000). However, to explore a medium that
contains all the main nutritional factors and obtain their
optimum levels is not an easy task. There is no general
defined medium for BD production by different microbial
strains because every strain has its own special nutritional
requirements depending on its environment. In our study,
statistically based experimental designs proved to be a
valuable tool in optimizing the medium for BD production
by the isolated strain K. pneumoniae SDM. Among the
eight variables tested by PB experiments, CSLP and
(NH4)2HPO4 were identified as the most important compo-
nents for BD production. Their optimal concentrations were
obtained by using statistical analysis of RSM.
(NH4)2HPO4 is the most important factor that influenced
the BD accumulation in the experiments as shown in
Table 3 (p<0.01). (NH4)2HPO4 offers the phosphorus
source as energy substance for cell growth and is also very
important to product formation. Garg and Jain (1995)
showed that phosphate stimulated diol production and
improved the conversion efficiencies. They proposed that
the effects on diol yields and glucose metabolism were due
to the effect of phosphate, which stimulated the complete
metabolism of the bacterium. Studies of Berbert-Molina et
al. (2001) also confirmed the significant role of
55
(NH4)2HPO4 as a source of nitrogen and phosphorous for
the production of BD.
CSLP, the solid state of corn steep liquor, a major by-
product of the corn wet-milling industry, contains approx-
imately 47% crude protein and is an inexpensive valuable
nutrient source available on a large scale (Parekh et al.
1999). It is commonly utilized in the fermentation industry
to produce a variety of substances, including the production
of ethanol by Zymomonas mobilis (Silveira et al. 2001),
protease by Streptomyces sp. (De Azeredo et al. 2006), and
lactic acid by Lactobacillus rhamnosus (Rivas et al. 2004).
The preliminary experiment results have clearly shown that
BD production was markedly enhanced by CSLP compared
with inorganic nitrogen source culture. This stimulating
effect was probably caused by certain essential microbial
nutrients present in CSLP (Kona et al. 2001), which
increased the cell concentration and stimulated BD produc-
tion because BD production is partially growth-associated
(Qin et al. 2006). Perhaps the complex nitrogen sources
(CSLP and (NH4)2HPO4) constitute better substrates for
BD production than simple nutritional components. In
microbial fermentations, the production costs are highly
dependent on the nitrogen source cost, as well as the carbon
source cost (Hofvendahl and Hahn-Hägerdal 2000). CSLP
is an alternative, low-cost, and high-yield media component
for BD fermentation and has an obvious economic
advantage.
The effects of different fermentation operation methods
such as batch, fed-batch, and continuous fermentation
operation modes on the fermentation of BD has been
studied (Syu 2001; Qin et al. 2006). Due to the inhibition
by high substrate concentration in batch fermentation, the
BD concentration and productivity were not high (Qin et al.
2006), which was the major disadvantage of the operation
56
mode. Continuous culture could generate high productivity
by allowing constant, usually stable, growth rates and
providing an essentially invariant microbial environment
(Ramachandran and Goma 1987), but the yield was
relatively low and increased the cost for the recovery of
BD, making the process less cost-efficient. Fed-batch
culture, which is generally superior to batch and continuous
processing (Lee et al. 1999) can avoid the effect of
substrate limitation and inhibition and control over the by-
products concentration and catabolite repression effects by
keeping the substrate concentration in the reactor below the
toxic level (Ezeji et al. 2004). It has been widely applied in
industry (Ko and Wang 2006). Most high BD production
has also been achieved using this culture mode (Qin et al.
2006). In this study, the batch fermentation phase was
firstly applied to study the kinetic parameters for growth of
K. pneumoniae SDM and understand its physiology in a
5-l B. Braun Biostat bioreactor. Then, the kinetic parame-
ters obtained from the batch data were used to determine an
optimal feed strategy to maximize BD yield for the fed-
batch fermentation. Different fed-batch feeding strategies
such as pulse fed batch, constant feed rate fed batch,
constant residual glucose concentration fed batch, and
exponential fed batch were introduced to determine the
optimal feed rate, residual glucose concentration, and
fermentation time to maximize BD productivity. Among
the four fed-batch feeding strategies, constant residual
glucose concentration fed batch had the maximum yield
and productivity (Table 7). Feed rate was designed
according to the glucose consumption rate in order to
maintain a glucose concentration of 20–30 g/l. In this way,
a high rate of BD synthesis was achieved. Compared with
other feed methods, this method may provide a more
suitable environment for the cell metabolism and BD
production. The cells grew rapidly and the exponential
phase was relatively short, only about 10 h. The cell
activity was different during the fermentation process
leading to major change of the glucose consumption rate
over the entire fermentation process: high in the exponen-
tial phase and decreasing gradually after that. This may be
the reason the other three methods were not as suitable for
the fermentation of BD.
In conclusion, we conducted a sequential statistical
experimental design to optimize the medium for BD
production by K. pneumoniae SDM. Then, the effect of
different fed-batch strategies on BD production was
studied. After the optimization, the BD yield increased to
150 g/l over a fermentation period of 38 h for the fed-batch
culture when using the optimized culture medium and the
diol productivity reached 4.21 g/l h. It was shown that
statistical experimental design offered an effect and feasible
approach for BD fermentation medium optimization. The
unusually high yield arose from three sources: the choice of
Appl Microbiol Biotechnol (2009) 82:49–57
a high-yield strain, optimization of the culture medium, and
use of an effective fermentation strategy. From an industrial
point of view, the optimum medium for BD production in
this study has simple components and is mainly composed
of common and cheap inorganic salts except for glucose
and a small quantity of CSLP, which is available for
efficient and economical production of BD on a large scale.
Acknowledgements This work was supported by the grants by
National Basic Research Program of China (2007CB707803) and
Chinese National Programs for High-Technology Research and
Development (2006AA02Z244 and 2006AA020101).
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