Bioprocess Biosyst Eng
DOI 10.1007/s00449-014-1348-5
ORIGINAL PAPER
Industrial vitamin B12 production by Pseudomonas denitrificans
using maltose syrup and corn steep liquor as the cost-effective
fermentation substrates
Wei Xia • Wei Chen • Wei-fu Peng •
Kun-tai Li
Received: 16 August 2014 / Accepted: 23 December 2014
Ó Springer-Verlag Berlin Heidelberg 2015
Abstract The aerobic Pseudomonas denitrificans is
widely used for industrial and commercial vitamin B12
fermentation, due to its higher productivity compared to
the anaerobic vitamin B12-producing microorganisms. This
paper aimed to develop a cost-effective fermentation
medium for industrial vitamin B12 production by P. deni-
trificans in 120,000-l fermenter. It was found that maltose
syrup (a low-cost syrup from corn starch by means of
enzymatic or acid hydrolysis) and corn steep liquor (CSL, a
by-product of starch industry) were greatly applicable to
vitamin B12 production by P. denitrificans. Under the
optimal fermentation medium performed by response sur-
face methodology, 198.27 ± 4.60 mg/l of vitamin B12
yield was obtained in 120,000-l fermenter, which was close
to the fermentation with the refined sucrose (198.80 mg/l)
and was obviously higher than that obtained under beet
molasses utilization (181.75 mg/l). Therefore, maltose
syrups and CSL were the efficient and economical sub-
strates for industrial vitamin B12 fermentation by
P. denitrificans.
Keywords Pseudomonas denitrificans  Vitamin B12 
Industrial fermentation  Maltose syrup  Corn steep liquor
Introduction
Vitamin B12 is used to describe the complex compounds of
cobalt corrinoid family, which is one of the most alluring
and fascinating molecules in the world of science and
W. Xia  W. Chen  W. Peng  K. Li (&)
Nanchang Key Laboratory of Applied Fermentation Technology,
Jiangxi Agricultural University, Nanchang 330045, China
e-mail: atai78@sina.com
medicine [1]. The biological forms of vitamin B12 include
adenosylcobalamin and methylcobalamin. As an important
growth factor for microorganisms and animals, vitamin B12
was first discovered as ‘‘the anti-pernicious anemia factor’’
in 1926 [2]. At present, vitamin B12 has been widely used
for the treatment of pernicious anemia and peripheral
neuritis [3], and it was found that vitamin B12 was the
essential cofactor for methionine synthase and (R)-meth-
ylmalonyl-CoA mutase in animals and humans [4].
Due to the chemical vitamin B12 synthesis processes being
highly complicated and costly, the commercial production of
vitamin B12 is exclusively via biosynthetic fermentation
processes. For microbial production of vitamin B12, there are
two different types, namely the aerobic and anaerobic fer-
mentation processes. By implementing the strategies of ran-
dom mutagenesis and genetic engineering, some industrial
microorganisms have been successfully exploited for the
commercial production of vitamin B12, such as Pseudomonas
denitrificans, Propionibacterium freudenreichii, Propioni-
bacterium shermanii and so on [5, 6]. As the typical vitamin
B12-producing anaerobes, Propionibacterium strains are
microaerophilic and their bioprocesses should be controlled
under very low oxygen concentrations [7]. Compared to the
anaerobic fermentation processes in Propionibacterium, the
aerobic P. denitrificans showed a more rapid cell growth and
efficient vitamin B12 productivity.
Our previous studies had been detailedly focused on the
industrial vitamin B12 fermentation processes by P. deni-
trificans in 120,000-l fermenter, in which a multi-stage
dissolved oxygen (DO) control strategy (20–48 h: 8–10 %;
49–106 h: 2–5 %; 107–168 h: below 2 %) [8], a pH-stat
control strategy (7.0–7.2) [9], and a continuous betaine
feeding strategy (5.0–7.0 g/l of betaine during 50–140 h of
fermentation) [10] were systematically established.
Although the vitamin B12 production was highly improved,
123

all of the above industrial fermentation medium utilized the
refined sucrose as the sole carbon source, resulting in high
medium costs. Alternatively, due to beet molasses (a by-
product of the sugar industry) containing a mass of ingre-
dients (such as sucrose, glutamate and betaine) favorable
for vitamin B12 production, a low-cost fermentation med-
ium was established with beet molasses as the main sub-
strate, and the fermentation cost was highly reduced [11].
However, if not being subjected to an acidification and heat
pretreatment, the undesirable components in beet molasses
would frequently result in an unstable fermentation process
by the inhibitory effect on cell growth of P. denitrificans,
such as coloring substances, heavy metals and unknown
compounds.
Based on this fact, the present work aimed to optimize
and develop an alternatively cost-effective fermentation
medium for the industrial vitamin B12 production using
maltose syrup, corn steep liquor (CSL), and betaine as the
main substrates.
Materials and methods
Microorganism and media
Pseudomonas denitrificans, an industrial strain donated by
Huarong Pharmacy Corporation (Shijiazhuang, China),
was used for vitamin B12 production, which was main-
tained on agar slant containing (g/l): sucrose, 30; peptone,
10; (NH4)2SO4, 0.25; (NH4)2HPO4, 1.5; MnSO4H2O, 0.1;
ZnSO47H2O, 0.1; agar, 20. The initial pH was adjusted to
7.2 with 1.0 M NaOH prior to sterilization.
Seed medium was composed of (g/l): sucrose, 30; CSL, 10;
KH2PO4, 5; (NH4)2SO4, 2.3; (NH4)2HPO4, 0.7; MnSO4H2O,
0.2; MgSO4, 1.5; ZnSO47H2O, 0.02; CoCl26H2O, 0.02; 5,6-
dimethylbenzimidazole (DMBI), 0.0045. The pH was adjus-
ted to 7.0–7.2 with 1.0 M NaOH prior to sterilization.
Original fermentation medium was as follows (g/l):
Maltose syrup (an enzymatic hydrolysis production from
corn starch, approximately 30.5 % (w/w) of total sugar
concentration), 260; CSL, 40; betaine, 20; (NH4)2SO4, 1;
MgSO4, 1.5; KH2PO4, 0.75; ZnSO47H2O, 0.08; CoCl2-
6H2O, 0.14; DMBI, 0.075. The pH was adjusted to 7.2–7.4
with 1.0 M NaOH before autoclaving.
Feed media (FM) for the fed-batch fermentation in
120,000-l fermenter were as follows (g/l): FM-a: maltose
syrup, 800; FM-b: betaine, 39; CoCl26H2O, 0.3; DMBI, 0.3.
Experimental design and statistical analysis
for fermentation medium optimization
The fermentation medium was optimized via response
surface analysis (RSM), in which a 23 full factorial central
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Bioprocess Biosyst Eng
composite designs (CCD) with nine replicates at the central
points were firstly performed using the statistical software
DPS v 3.01, and then the statistical analysis of results was
accomplished by R2.13.1 software.
Fermentation in shake flasks
Preculture was carried out in a 250-ml Erlenmeyer flask
containing 40 ml of seed medium inoculated with cells
from fresh slant, and cultivated at 30 °C on a rotary shaker
at 180 rpm for 20 h. The seed culture was then transferred
to a 250-ml Erlenmeyer flask containing 30 ml fermenta-
tion medium with 10 % inoculum, and incubated at 32 °C
on a rotary shaker at 180 rpm for 120 h.
Fermentation in 120,000-l fermenter
Three-stage fermentation was performed in 120,000-l fer-
menter, including two stages of seed growth and one stage
of vitamin B12 production. The first-stage culture was
carried out in a 150-l seed fermenter containing 100 l
sterile medium with inoculum from eight fresh slants,
which was implemented at 28 °C for 40 h with an aeration
rate at 0.5 v/v/min and an agitation speed at 180 rpm. Then
the primary seed culture (80–90 l) was transferred into a
9,000-l secondary seed fermenter with 5,000 l of seed
medium. It was cultivated at 28 °C with stirrer speed at
130 rpm and aeration rate at 0.4 v/v/min for 30 h. Then the
second seed culture (about 5 % of the final working vol-
ume) was transferred into the 120,000-l fermenter con-
taining 75,000 l of fermentation medium. A fed-batch
fermentation was performed, which was controlled at
32 °C and finished at 180 h.
The 120,000-fermenter was equipped with three 3-bla-
ded propeller impellers, a temperature probe, a pH probe
(Mettler Toledo) and a dissolved oxygen (DO) probe
(Mettler Toledo). The oxygen uptake rate (OUR) and
carbon dioxide evolution rate (CER) were collected online,
which were calculated by the analysis of the inlet and
exhaust gases.
Analytical methods
Vitamin B12 concentration in fermentation broth was
determined by HPLC. Broth sample (25 ml), to which
2.5 ml of NaNO2 8 % (w/v) and 2.5 ml of glacial acetic
acid were added, were boiled for 30 min. The mixture was
then filtrated, and 20 ll of NaCN 10 % (w/v) was added to
1 ml of aqueous phase. The resulting upper aqueous phase
was injected into the HP1100 HPLC system (Agilent).
Hypersil APS-2 NH2 column (4.6 mm 9 250 mm, 5 lm)
was employed for HPLC analysis with a flow rate of
1.7 ml/min, a wavelength of 360 nm, and a column
Bioprocess Biosyst Eng
temperature of 30 °C. The mobile phase was 250 mmol of
phosphoric acid/acetonitrile, (30/70, v/v).
Cell biomass was quantified with dry cell weight
(DCW). The centrifuge tube (50 ml) equipped with 30 ml
of the culture broth was centrifuged at 5,000 rpm for
10 min. After washing twice with distilled water, the cell
precipitate was dried to a constant weight at 105 °C, and
then was converted to the standardized DCW (g/l).
The total sugar concentrations of the fermentation broths
carried out in shake flasks and industrial fermenter were
determined by the dinitrosalicylic acid reagent (DNS)
method [12]. According to the method described by Niu
et al. [13], a HPLC system (Waters 2695) equipped with an
Alltech 2000 Evaporative Ligh Scattering detecor (Alltech
Associates, Deerfield, USA) was used to measure the
concentrations of maltose and glucose in corn starch syr-
ups, and the conditions of HPLC were as follows: Column,
Hypersil NH2 (4.6 mm 9 250 mm, 5 lm); mobile phase,
acetonitrile/water = 70/30 (v/v); flow rate, 1.2 ml/min;
column temperature, 30 °C; inject volume 10 ll.
Amino nitrogen content in the broth was quantified by
the formaldehyde titration method [14].
The categories and concentrations of amino acids in
CLS were assayed according to the method reported by
Ebert [15].
Results and discussion
Effects of glucose syrup and maltose syrup
on P. denitrificans fermentation
Carbon source is one of the main nutritional components
involved in a fermentation medium, but the usual refined
sugars (e.g. glucose, sucrose and maltose) are uneconomical
to an industrial fermentation. Compared to the refined sug-
ars, a range of low-cost syrups (such as glucose syrup and
maltose syrup) can be produced from corn starch by means
of enzymatic or acid hydrolysis [16]. To investigate whether
the corn starch syrups could replace the refined sucrose for
vitamin B12 production, the P. denitrificans fermentation
processes with glucose syrup and maltose syrup as the car-
bon sources were, respectively, carried out in shake flasks,
and Fig. 1 showed the kinetics of cell growth, total sugar
consumption, pH and vitamin B12 biosynthesis.
As shown in Fig. 1a, compared with glucose syrup as
the carbon source, the cell growth under maltose syrup was
significantly higher during the whole fermentation pro-
cesses. In consistent with the higher consumption rate of
total sugar (Fig. 1b), it was observed that the pH values
under glucose syrup were obviously lower than the fer-
mentation with maltose syrup as the carbon source, as
shown in Fig. 1c. During the whole fermentation
processes, the pH under maltose syrup could maintain at a
stable range (approximately 6.50), while glucose syrup
would cause the broth pH sharply decrease to 5.40–5.50 at
96–120 h of fermentation. As a result, the obtained vitamin
B12 production under maltose syrup was significantly
higher than that using glucose syrup, which were finally
achieved at 66.74 ± 2.56 g/l and 51.32 ± 0.81 mg/l,
respectively, as shown in Fig. 1d.
The above results revealed that maltose syrup could be
acted as an alternative applicable carbon source for vitamin
B12 production by P. denitrificans, which would remark-
ably reduce the cost of carbon source.
The analysis of amino acids compositions in CSL
Besides carbon source, nitrogen source also plays an
important role in the microbial fermentation, and a cost-
effective organic nitrogen source becomes the preferred
medium ingredient to substitute for the complex nitrogen
sources (such as yeast extract and peptone). CSL, a by-
product of starch industry, is widely used in microbial
fermentation, due to containing a rich complement of
important nutrients such as amino acids, vitamins, biotins
and trace elements [17, 18].
To explore whether CSL being applicable to vitamin B12
production by P. denitrificans, the amino acids compositions
in CSL (Hebei Zhongying Starch Co., Ltd., China) were
analyzed. As shown in Fig. 2, the CSL included eighteen
different kinds of amino acids, and the total concentration of
these amino acids was up to 23.093 mg per 100 mg of CSL,
which demonstrated that the CSL would be an appropriate
organic nitrogen source for microbial fermentation.
During vitamin B12 biosynthetic process, its tetrapyrrole
structure starts from the C-5 skeleton of glutamate [19]. Our
earlier research had also clearly illustrated that an enriched
glutamate in fermentation medium would be in favor of cell
growth and vitamin B12 production by P. denitrificans [11].
Consequently, with regard to the preferential organic nitrogen
source for vitamin B12 fermentation, the embedded glutamate
concentration is one of the crucial factors worth considering. It
was noteworthy that glutamate was the second majority
component (only lower than proline) contained in the CSL,
which concentration could reach 3.539 ± 0.163 mg/100 mg
CSL, as shown in Fig. 2. Therefore, the amino acids compo-
sitions in the CSL implied that the CSL would potentially be
an efficient and low-cost organic nitrogen source for vitamin
B12 fermentation by P. denitrificans.
Optimization of fermentation medium with maltose
syrup and CSL as the main substrates
Except the carbon and nitrogen sources being essential to
microbial fermentation, the exogenous addition of
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 Bioprocess Biosyst Eng

(a) 25 (b) 90
80

Concentration of total sugar (g/l)

20
70
60
DCW (g/l)

15
50
40
10
30
Glucose syrup
5 Maltose syrup 20 Glucose syrup
Maltose syrup
10
0 0
0 24 48 72 96 120 0 24 48 72 96 120
Time (h) Time (h)

(c) 7.0 70
(d)
6.5 60
Glucose syrup
Maltose syrup
50
6.0
(mg/L)
40
5.5
pH

12
Vitamin B

30
5.0 Glucose syrup
20
Maltose syrup
4.5
10

4.0 0
0 24 48 72 96 120 72 96 120
Time (h) Time (h)

Fig. 1 Effects of glucose syrup and maltose syrup on the cell growth (a), pH (b) and vitamin B12 production (c) during P. denitrificans shake-
flask cultivation. Values represent the averages of three measurements and error bars indicate standard deviation

6.0 precursors and production promoters can strongly regulate

Concentrations of amino acid (mg/100mg)

5.5 and facilitate the biosynthesis of target metabolites. During

5.0 vitamin B12 biosynthetic pathway in P. denitrificans, there
4.5 are eight methylation reactions catalyzed by six different
4.0 methyltransferases, and betaine (N,N,N-trimethylglycine)
3.5
is widely chosen as the typical methyl-group donor for
3.0
vitamin B12 production [20–22], due to three methyl
2.5
groups involved in their molecules.
2.0
Based on this fact, the three medium components
1.5
1.0
(maltose syrup, CSL and betaine) were further optimized
0.5 for vitamin B12 fermentation using response surface
0.0 methodology (RSM), an effective and successful experi-
Trp Cys Met Tyr Ile His Lys Phe Thr Ser Gly Arg Val Ala Asp Leu Glu Pro ment design procedure [23, 24]. The levels and actual
Amino acid consisted in corn steep liquor values of the above three variable factors and experimental
Fig. 2 Concentrations of amino acids contained in corn steep liquor.
results (vitamin B12 yield) were presented in Table 1, and
Values represent the averages of three measurements and error bars the corresponding regression analysis accomplished by
indicate standard deviation R2.13.1 software was summarized in Table 2.

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Bioprocess Biosyst Eng

From the results of statistical analysis shown in Table 2, Table 2 Results of the regression analysis of the central composite
it was observed that the significant terms (a = 0.05) were design
as follows: X1, X2, X3, X21, X22, X23 and X1 9 X3. The fol- Term Estimate t value Pr ([|t|)
lowing second-order model for predicting vitamin B12
Intercept -397.500 -4.241 0.000823**
yield (Y) was established:
X1 2.084 3.126 0.007443**
Y ðmg/LÞ ¼ 397:500 þ 2:084X1 þ 5:070X2 þ 7:241X3 X2 5.070 4.867 0.000249**
 0:003723X12  0:01722X22  0:05079X32  0:004338 X1 X3 7.241 7.343 0.00000366**
X1 9 X1 -0.003723 -2.973 0.010071*
 X2  0:1003 X1  X3
X2 9 X2 -0.01722 -3.439 0.003989**
This regression model for vitamin B12 production was X3 9 X3 -0.05079 -2.536 0.023773*
highly significant (P \ 0.0001) with a satisfactory value of X1 9 X2 -0.004338 -1.229 0.239289
determination coefficient (R2 = 0.9567), which indicated X1 9 X3 -0.1003 -7.105 0.00000529**
that the second-order model was adequate for the results 2
R = 0.9567, F = 38.65, Prob [ F = 0.0001
obtained in experiments. Figure 3 showed the resulting * and ** indicate significance at 0.05 and 0.01 level, respectively
response surface concerning the effects of maltose syrup,
CSL and betaine on the vitamin B12 production, and the be obtained as follows: X1 = 251.29 g/l, X2 = 49.07 g/l,
response surface had a maximum point. It was found that X3 = 22.83 g/l. Under the optimal fermentation medium,
the interaction between maltose syrup and betaine was the theoretical maximum of vitamin B12 yield was
significant, which revealed that the concentrations of 71.42 mg/l. Five replicated experiments were further per-
maltose syrup and betaine in fermentation medium would formed to validate the second-order model at the optimal
be crucial to vitamin B12 production. point of maltose syrup, CSL and betaine, and the obtained
Based on the equations derived by differentiation of the vitamin B12 production was 72.55 ± 1.08 mg/l (71.15,
second-order model, the optimal values of the model could 71.88, 72.63, 73.14 and 74.94 mg/l, respectively), which

Table 1 Experimental design No. X1 (maltose syrup) X2 (corn steep liquor) X3 (betaine) Vitamin B12 (mg/l)
and results of central composite
design Coded Real (g/l) Coded Real (g/l) Coded Real (g/l)

1 1 280 1 50 1 25 69.76
2 1 280 1 50 -1 15 65.83
3 1 280 -1 30 1 25 69.82
4 1 280 -1 30 -1 15 45.49
5 -1 240 1 50 1 25 72.82
6 -1 240 1 50 -1 15 68.25
7 -1 240 -1 30 1 25 69.07
8 -1 240 -1 30 -1 15 44.78
9 -1.68179 226.364 0 40 0 20 62.92
10 1.68179 293.636 0 40 0 20 61.2
11 0 260 -1.68179 23.18 0 20 52.86
12 0 260 1.68179 56.82 0 20 69.94
13 0 260 0 40 -1.68179 11.59 55.35
14 0 260 0 40 1.68179 28.41 70.01
15 0 260 0 40 0 20 69.5
16 0 260 0 40 0 20 67.15
17 0 260 0 40 0 20 65.55
18 0 260 0 40 0 20 68.7
19 0 260 0 40 0 20 67.39
20 0 260 0 40 0 20 65.65
21 0 260 0 40 0 20 65.19
22 0 260 0 40 0 20 68.91
23 0 260 0 40 0 20 66.27

123

Fig. 3 Three-dimensional presentation of the response surface for the concentrations of maltose syrup, betaine and corn steep liquor on vitamin
B12 yield
was close to the optimum value predicted by the second-
order model.
Industrial vitamin B12 fermentation with the optimized
medium
The industrial vitamin B12 fermentation was performed
using the optimized fermentation medium in a 120,000-l
fermenter, in which the fermentation conditions were
according to our previous control strategies [8]. Figure 4
presented the typical profiles of the online fermentation
parameters, such as agitation speed, air flow, DO, OUR,
CER and pH.
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Bioprocess Biosyst Eng
From Fig. 4, the agitation speed and air flow were
controlled at 78 rpm and 850–1100 m3/h, respectively.
Under this kind of oxygen supply strategy, the DO was
sharply decreased to below 2 % at 30 h, and then main-
tained at this level until the end of fermentation. Accom-
panied with the increase of the microbial respiration
strength, the OUR and CER presented a significantly
increasing tendency during 0–30 h. Due to the DO con-
centration entering into a limiting level, the OUR and CER
values almost remained at a constant level. Our previous
research had proved that a limiting DO concentration was
favorable for vitamin B12 biosynthesis, especially in the
later fermentation stages [8]. Therefore, a two-stage control
Bioprocess Biosyst Eng
Fig. 4 Profiles of the online
fermentation parameters
(Agitation rate; Air flow; pH;
Agitation rate (rpm); Air flow (20*m3/h)
Dissolved oxygen, DO; Oxygen
DO(%); OUR, CER (mmol/l/h);
uptake rate, OUR; Carbon
dioxide evolution rate, CER)
with maltose syrup and corn
steep liquor as the main
substrates for P. denitrificans
cultivation in a 120,000-l
fermenter
strategy on air flow was implemented during industrial
vitamin B12 fermentation processes. However, once the air
flow was dropped from approximately 1,100–850 m3/h at
104 h, the limiting DO concentration would result in an
immediate decrease of OUR and CER, and this kind of
phenomenon was identical to the vitamin B12 fermentation
process implemented by Wang et al. [25]. Without any acid
or alkali exogenously added to the fermentation broth, the
pH values could stably keep at 7.0–7.30 during the whole
fermentation processes, which was the most beneficial
range to the cell growth and vitamin B12 biosynthesis by
P.denitrificans [9].
Figure 5a showed the kinetics of total sugar and amino
nitrogen during the fermentation processes in 120,000-l
fermenter. Under the utilization of the optimal fermenta-
tion medium, the initial concentrations of total sugar and
amino nitrogen in broth could achieve at 76.4 ± 2.6 g/l
and 1050 ± 30 mg/l, respectively. Along with the fer-
mentation process, the sugar consumption rate presented an
accelerated trend, resulting in the total sugar concentration
rapidly decreasing to 34.8 ± 0.9 g/l at 42 h. At this
moment, the maltose syrup (FM-a) was continuously fed to
the fermentation broth, to maintain the total sugar among
30–35 g/l. However, the amino nitrogen concentration
presented an obvious three-stage kinetics: a rapid decrease
before 48 h, a stable status (about 400 mg/l) during
48–144 h, and a drastic rise due to cell autolysis after
144 h.
Figure 5b summarized the time courses of biomass
growth and vitamin B12 production under the optimal fer-
mentation medium. In consistent with the kinetics of DO,
OUR, CER and total sugar, a rapid uptrend of cell growth
90 9.0
8.5
80
70 8.0
Agitation rate
pH
7.5
60
7.0
50 CER Air flow
pH
6.5
40
6.0
OUR
30
5.5
20 5.0
DO
10 4.5
0 4.0
0 12 24 36 48 60 72 84 96 108120132144156168180
Time (h)
was observed during 0–60 h, which revealed that the
maltose syrup and CSL as the main medium substrates
were greatly propitious to the cell growth of P. denitrifi-
cans. Simultaneously, the vitamin B12 yield also presented
a rapid increase under the utilization of this fermentation
medium. As a result, the maximal vitamin B12 was up to
198.27 ± 4.60 mg/l at the end of fermentation (180 h),
which was close to the industrial vitamin B12 production
under using the refined sucrose (198.80 mg/l of final vita-
min B12 yield) [8], and was obviously higher than that
obtained with beet molasses as the main fermentation
medium (181.75 mg/l of final vitamin B12 yield) [11].
Many microorganisms have the capacity to biosynthe-
size vitamin B12, such as P. denitrificans, Salmonella ty-
phimurium, Propionibacterium freudenreichii,
Propionibacterium shermanii, Bacillus megaterium, Rho-
dopseudomonas protamicus, Rhizobium cobalaminogenum
and Streptomyces olivaceus [6]. Among these microor-
ganisms, P. denitrificans is the most efficient vitamin B12-
producing strain, and it is conceivable that its productivity
may reach 300 mg/l [1]. Although a significant improve-
ment of vitamin B12 production had been achieved by
means of random mutagenesis, genetic engineering and
metabolic engineering of P. denitrificans, a problem wor-
thy of attention was how to design an optimal cost-effec-
tive fermentation medium for industrial vitamin B12
production, because the medium cost generally occupied
almost 50 % of most fermentation products [26]. It was
well known that beet molasses was the most widely used
carbon source for industrial vitamin B12 fermentation by P.
denitrificans. However, the undesirable components in beet
molasses would cause an unstable vitamin B12
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 Bioprocess Biosyst Eng

Fig. 5 Time course of total (a) 100 1200

sugar (a), amino nitrogen (a),
cell growth (b) and vitamin B12 90 1100

Concentration of amino nitrogen (mg/l)

production (b) with maltose

Concentration of total sugar (g/l)

syrup and corn steep liquor as 80 1000
the main substrates for P. Total sugar
70 900
denitrificans cultivation in a Amino nitrogen
120,000-l fermenter. Values 60 800
represent the averages of three
measurements and error bars 50 700
indicate standard deviation
40 600
30 500
20 400
10 300
0 200
0 12 24 36 48 60 72 84 96 108 120 132 144 156 168 180
Time (h)

(b) 35 240

30 200

25
160

Vitamin B12(mg/l)
DCW (g/l)

20
120
15
80
10
DCW
Vitamin B12 40
5

0 0
0 12 24 36 48 60 72 84 96 108120132144156168180
Time (h)

fermentation process. As a refined sugar, sucrose could
avoid this situation, but its high price would make indus-
trial vitamin B12 fermentation become uneconomical.
Therefore, it was valuable to develop an alternatively cost-
effective fermentation medium for the industrial vitamin
B12 production.
The present work was a first report on the utilization of
maltose syrup and CSL for industrial vitamin B12 produc-
tion. On the calculation of pure sugar, the price of maltose
syrup was approximately 53 % of sucrose, but 136 % of
beet molasses. However, an extra of about 20 g/l sucrose
should be added to the fermentation medium when using
beet molasses for vitamin B12 fermentation [11]. Similar to
the refined sucrose, the maltose syrup could also effectively
avoid an unstable vitamin B12 fermentation process caused
by the undesirable components in beet molasses. There-
fore, compared to beet molasses, maltose syrup displayed
more competitive advantage for vitamin B12 production. In
a word, the developed fermentation medium with maltose
syrup and CSL as the main substrates were efficient and
economical to industrial vitamin B12 production by P.
denitrificans in 120,000-l fermenter.
Conclusions
Based on investigating the effects of maltose syrup on P.
denitrificans fermentation process, together with analyzing
the amino acids compositions in the CSL, this paper suc-
cessfully established a cost-effective fermentation medium

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Bioprocess Biosyst Eng
for industrial vitamin B12 production by P. denitrificans in
a 120,000-l fermenter, with maltose syrup and corn steep
liquor as the main substrates. As a result, the industrial
vitamin B12 yield could be achieved at 198.27 ± 4.60 mg/
l.
Acknowledgments This work was financially supported by the
National Natural Science Foundation of China (Grant No. 31460019),
Jiangxi Key Project of Basic Research Program (20143ACB21005),
Training Program for Young Scientists of Jiangxi Provincial
Department of Science and Technology (20142BCB23025), and
International Scientific and Technological Cooperation Projects of
Jiangxi Provincial Department of Science and Technology
(20141BDH80033). We are also grateful to Shijiazhuang Pharma
Group Hua Rong Pharmaceutical Co. Ltd for supporting this work.
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