Cao et al.

Bioresources and Bioprocessing (2015) 2:9

DOI 10.1186/s40643-014-0032-6

RESEARCH Open Access

Improving the stability of glutamate fermentation

by Corynebacterium glutamicum via supplementing
sorbitol or glycerol
Yan Cao1*, Zhen-ni He2, Zhong-ping Shi2 and Mpofu Enock3

Abstract
Background: Corynebacterium glutamicum is widely used in glutamate fermentation. The fermentation feature of
the strain varies sometimes. These variations may lead to the reduction in the ability of the strain to resist
environmental changes and to synthesize glutamate, resulting in abnormal glutamate fermentations.
Results: In the abnormal glutamate fermentations, glutamate accumulation stopped after glucose feeding and the
final glutamate concentration was at a lower level (50 to 60 g/L). The rNAD+/rNADH ratio was lower than that in
normal batch which was reflected by lower oxidation-reduction potential (ORP) value. The abnormal fermentation
performance was improved when glucose was co-fed with sorbitol/glycerol at a weight ratio of 5:1 or adding 10 to
15 g/L of sorbitol/glycerol in the initial medium. Under these conditions, glutamate synthesis continued after substrate
(s) feeding and final glutamate concentration was restored to normal levels (≥72 g/L). r+NAD/rNADH ratio, ORP, and pyruvate
dehydrogenase (PDH), isocitrate dehydrogenase (ICDH), and cytochrome c oxidase (CcO) activities were maintained
at higher levels.
Conclusions: Sorbitol and glycerol were not used as carbon sources for the fermentation. They were considered
as effective protective agents to increase cells' resistance ability against environmental changes and maintain
key enzymes activities.
Keywords: Enzyme activity; Fermentative stability; Glutamate fermentation; Oxidation-reduction potential;
Protective substance; rNAD+/rNADH

Background optimal value at different stages during glutamate fer-

L-Glutamate is mainly used as a flavor enhancer in food in- mentation usually varied [3]. The metabolic flux dis-
dustry and nutrient in pharmaceutical industry. The annual tribution can be altered by variation of NAD+/NADH
production has exceeded 2.2 million tons by fermentation ratio or rNAD+/rNADH ratio in glutamate fermentation [4].
with Corynebacterium glutamicum [1]. The biosynthetic NAD+/NADH ratio is indirectly reflected by oxidation-
pathway of glutamate includes complex enzymatic reac- reduction potential (ORP) [5,6], which represented the
tions, as shown in Figure 1a. The enzymes effectively con- redox state of the cells. The optimal ORP range correspond-
vert substrate (such as glucose) into glutamate only when ing to different fermentation processes is different. For ex-
they are well coordinated with coenzymes NAD+ or ample, maximum lysine yield was obtained when ORP was
NADH. Intracellular levels of NAD+ and NADH signifi- controlled between the range of −230 and −210 mV, while
cantly affect the catalytic efficiency of the enzymes [2]. the preferable ORP range was −275 to −225 mV in homo-
The ratio of NAD+/NADH in vivo is a key factor affecting serine and valine fermentations [6,7]. The redox state
energy transfer and redox state of the cells, and the of cells is changed if certain auxiliary substances (such
as sorbitol and glycerol) are supplemented and intracellu-
lar NAD+/NADH ratio can be varied correspondingly
* Correspondence: [email protected] [8]. These auxiliary substrates are usually non-repressive
1
National University of Singapore (Suzhou) Research Institute, 377 Linquan
Street, Suzhou, Jiangsu Province, China
Full list of author information is available at the end of the article

© 2015 Cao et al.; licensee Springer. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction
in any medium, provided the original work is properly cited.
Cao et al. Bioresources and Bioprocessing (2015) 2:9 Page 2 of 11

Figure 1 Simplified metabolic pathway of glutamate synthesis and generation/consumption of NAD+ and NADH. (a) Metabolic pathway
of glutamate synthesis; (b) simplified pathway of generation/consumption of NAD+ and NADH. GLC, glucose; PYR, pyruvate; Ac-CoA,
acetyl coenzyme A; ICIT, isocitrate; α-KG, α-ketoglutarate; SUC, succinate; MAL, malate; OAA, oxaloacetate; PDH, pyruvate dehydrogenase;
PC, pyruvate carboxylase; ICDH, isocitrate dehydrogrnase; ODHC, α-ketoglutarate dehydrogenase; GDH, glutamate dehydrogenase; CcO,
cytochrome c oxidase.

carbon sources. They can protect cells against stress in feeding mixed substrates has been applied in other fermen-
their living environment, enhance cell viability, and reduce tations. This was an effective method to decrease cell mor-
the metabolic burden [9-12]. It was reported that the pro- tality, maintain the enzyme activity, and promote targeted
duction of alkaline polygalacturonate lyase and lipase in- metabolite production [9-11]. Similar studies with regard
creased by 1.85-fold and 8.7-fold, respectively, when the to glutamate fermentation are also important, but few have
strategy of methanol/sorbitol co-feeding was adopted been reported. In this study, sorbitol or glycerol was either
[10,13]. Arruda and Felipe found that xylitol productivity co-fed with glucose or added in the initial medium, aiming
could be increased by 35% when glycerol was added in the at protecting the cells against environmental change/stress
medium [14]. Therefore, fermentation with mixed carbon and stabilizing glutamate productions. Meanwhile, the the-
sources was considered as an effective way to enhance the oretical mechanism was interpreted. The results gained in
targeted metabolite productions. this study will provide some useful information and refer-
C. glutamicum used in industry is usually stored at ence to the glutamate fermentation industry in terms of
4°C for a short time and replaced regularly. In this way, stabilizing the glutamate production.
production fluctuation resulting from strain change can be
avoided. However, sometimes the fermentation characteris- Methods
tics of the strain vary, resulting in decreased glutamate syn- Strain and culture condition
thesis ability and resistance to environmental changes. In C. glutamicum ATCC13032 was used in this study. The
such a case, fermentation performance becomes abnormal seed microorganism was grown in a shaker at 32°C and
and glutamate production also ends at a very low level. 200 r/min for 8 to 10 h in liquid medium containing
Glutamate production fluctuated in a large range, and fer- (in g/L) glucose 25, K2HPO4 1.5, MgSO4 0.6, MnSO4
mentation stability decreased greatly. Frequent rejuven- 0.005, FeSO4 0.005, corn slurry 25, and urea 2.5 (separ-
ation is a common solution to this problem. But it is a ately sterilized). Initial pH was adjusted to 7.0 to 7.2.
costly, time-consuming, and troublesome procedure. Fur- The medium for jar fermentation contained (in g/L)
thermore, glutamate is a low value-added product. It is glucose 140, K2HPO4 1.0, MgSO4 0.6, MnSO4 0.002,
more economical to adopt a simple way with low operation FeSO4 0.002, thiamine 5.0 × 10−5, corn slurry 15, and
cost to maintain the fermentative stability. The strategy of urea 3.0 (separately sterilized).
Cao et al. Bioresources and Bioprocessing (2015) 2:9 Page 3 of 11

The fed-batch fermentation was implemented in a 5-L carbon sources (18 h) was set as the unit (1), and REA
fermentor (BIOTECH-5BG, Baoxing Co., Shanghai, China) after feeding glucose/mixed carbon sources (26 h) was
equipped with on-line DO/pH/ORP electrodes. Initial described by Eq. (1).
medium volume was 3 L, and air aeration rate was
1.33 vvm. The temperature was maintained at 32°C dur- E aft ðk Þ
REAaft ðk Þ ¼ ð1Þ
ing the entire fermentation period (about 34 to 36 h). pH E bef ðk Þ
was maintained at 7.0 to 7.2 by automatically pumping in
25% (w/v) ammonia water. Dissolved oxygen (DO) was Where Ebef(k) and Eaft(k) referred to the activities of
controlled at 20% of air saturation by manually adjusting k-th enzyme (PDH, ICDH, and CcO) before and after
agitation rate. Concentrated glucose (50%, w/v) was fed glucose/mixed carbon sources feeding.
when glucose concentration was lower than 20 g/L. Sorb-
itol or glycerol was supplemented by the following two Modeling and calculation of rNAD+/rNADH ratio
methods: The glutamate synthesis pathway was depicted according to
the map reported [16], shown in Figure 1a. NADH and
Method #1: 50% (w/v) sorbitol or glycerol solution was NAD+ were generated or consumed to run the entire fer-
co-fed with the addition of concentrated glucose solution. mentation, and they were closely associated with glucose
The feeding ratio of 1:5 (w/w) sorbitol/glycerol versus consumption, glutamate synthesis, CO2 release, and O2 con-
glucose was applied. sumption rates. Therefore, the generation or consumption
Method #2: 5 to 15 g /L sorbitol or glycerol was added rates of NADH and NAD+ could be determined by a couple
into the initial medium before inoculation. Only of measurable reaction rates, such as glucose consumption
glucose was fed when glucose concentration was lower rate (rGLC), glutamate formation rate (rGLU), CER, and OUR.
than 20 g/L. The metabolic pathways of NADH and NAD+ were simpli-
fied as Figure 1b based on the following assumptions:
Analytical and measurement methods
(1)The main products were glutamate and CO2, because
Cell concentration was assayed by spectrophotometer at
the concentrations of other byproducts (lactate,
620 nm (OD620). Glucose and glutamate concentrations
acetate, and other amino acids) were very low.
were measured by a biosensor (SBA-40C, Shandong
(2)Pentose phosphate (PP) pathway was ignored because
Science Academy, Jinan, China). The concentrations of
it was not related to generation/consumption of
sorbitol and glycerol were analyzed by HPLC (Hitachi
NADH or NAD+.
Chromaster Organizer, Hitachi, Ltd, Chiyoda-ku, Japan)
(3)The metabolic flux into the two reaction branches at
equipped with an ion exclusion column (Aminex HPX-87H,
pyruvate node followed the ideal condition, namely
300 mm × 7.8 mm, Bio-Rad, Hercules, CA, USA) and a dif-
r2 = r3 in Figure 1b [16]. Therefore, the glyoxylate
ferential refractive index detector at 30°C. The mobile phase
shuttle was ignored.
was 0.005 mol/L H2SO4 at a flow rate of 0.6 mL/min [10].
(4)The intermediate carbon metabolites were in
Two electronic balances (JA1102, Haikang Co., Shanghai,
pseudo-steady-state, and the net accumulation of
China) were connected to the computer and used to moni-
them was 0. But it was not applied for NADH and
tor the feeding amount of glucose and sorbitol or glycerol
NAD+. rNAD+/rNADH ratio was closely and positively
solution. O2 and CO2 partial pressure in exhaust gas were
associated with the NAD+/NADH ratio, while rNAD+
on-line measured by a gas analyzer (LKM2000, Lokas Co.,
actually represented NADH consumption rate
Daejeon, Korea). CO2 evolution rate (CER), O2 uptake rate
(r(C)NADH) and rNADH represented NADH formation
(OUR), and respiratory quotient (RQ) were then calculated
rate (r(F)NADH). NADH formation rate (r(F)NADH)
by standard formula.
could differ with its consumption rate (r(C)NADH), which
led the variation in rNAD+/rNADH ratio.
Enzyme activity assay (5)Glutamate fermentation was a non-growth associated
The activities of pyruvate dehydrogenase (PDH) and iso- process; the cell concentration in production phase
citrate dehydrogenase (ICDH) were analyzed by the basically stayed at a constant level or declined slightly.
methods reported [15,16]. Cytochrome c oxidase (CcO) So, we used the volume reaction rate to replace the
was assayed using the kit for bacteria (Genmed Scientifics specific reaction rate for convenience purpose.
Inc., Wilmington, DE, USA). The enzyme activity was
expressed as U/mg-DCW, where 1 U was the quantity The simplified metabolic pathway (Figure 1b) contains
of dry cell converting 1 μmol NAD+ per minute. The rela- nine reactions shown in the Appendix, which covers the
tive enzymatic activity (REA) was used for comparison basic reactions occurring in EMP pathway, tricarboxylic
and interpretation. REA before feeding glucose/mixed acid (TCA) cycle, CO2 fixing reaction, respiratory chain,
Cao et al. Bioresources and Bioprocessing (2015) 2:9 Page 4 of 11

and glutamate synthesis. According to the assumptions Where rFNAD+(t) is the NAD+ formation rate (mmol/L/h),
and simplifications above, the rates of all the reactions rFNADH(t) NADH formation rate (mmol/L/h), rU O2(t) O2
were coupled as follows. uptake rate (mmol/L/h), rFCO2(t) CO2 evolution rate
(mmol/L/h), rUCO2(t) CO2 uptake rate (mmol/L/h), rGLC(t)
r 1 ¼ r GLC ð2Þ glucose consumption rate (mmol/L/h), and rGLU(t) glutam-
r 2 ¼ r 3 ¼ r 4 ¼ r 5 ¼ 0:5r 1 ð3Þ ate production rate (mmol/L/h).

r 6 ¼ r 7 ¼ r 5 −r 8 ð4Þ
Results and discussion
r 8 ¼ r GLU ð5Þ
Fermentation performance of normal and abnormal
r 9 ¼ 2r O2 ¼ 2 OUR ð6Þ batches
Final glutamate production and cell concentration were
The generation/consumption rates of NADH and NAD+ two factors reflecting the fermentation performance. In
as well as rNAD+/rNADH ratio at a specified time t could be ‘normal’ fermentation, final glutamate concentration and
calculated by the equations of (7) to (16). the maximum cell concentration (OD620) were more than
70 g/L and 50, respectively. Otherwise, fermentations were
categorized into ‘abnormal.’ Glutamate fermentation was
r F1
NADH ðt Þ ¼ 2r 1 ðt Þ ¼ 2r GLC ðt Þ ð7Þ non-growth associated, and glutamate was accumulated
when cell growth had almost ceased (after 10 h). At this
r F2 F1
NADH ðt Þ ¼ r 2 ðt Þ ¼ r CO2 ðt Þ ð8Þ moment, about 1/3 glucose in the initial medium was con-
sumed. Additional glucose was supplemented during the
main glutamate production phase at 18 to 20 h. In the
r F3 F2
NADH ðt Þ ¼ r 5 ðt Þ ¼ r CO2 ðt Þ ð9Þ normal batch, glutamate concentration still increased after
glucose was fed, although glutamate accumulation rate be-
came slow. However, glutamate production stopped after
r F4 F3
NADH ðt Þ ¼ r 6 ðt Þ ¼ r CO2 ðt Þ ð10Þ glucose feeding in the abnormal batch, and the final glu-
tamate concentration was around 50 g/L. In this case, cell
growth and glutamate production before glucose feeding
r F5
NADH ðt Þ ¼ r 7 ðt Þ ¼ r 6 ðt Þ ð11Þ
were almost the same as those in the normal fermentation
(Figure 2a,b). There was no significant difference in the
r F1
NADþ ðt Þ ¼ r GLU ðt Þ ð12Þ changing trend of RQ between the normal and abnormal
batches. However, the changing pattern of ORP in the ab-
normal batch differed from that of normal batch signifi-
r F2 U
NADþ ðt Þ ¼ r 9 ðt Þ ¼ 2r O2 ðt Þ ¼ 2OURðt Þ ð13Þ
cantly (Figure 2c,d). ORP was maintained in the normal
range (−75 to −85 mV) before 20 h (glucose was fed at
rU
CO2 ðt Þ ¼ r 3 ðt Þ ð14Þ 18 h) and decreased slowly to a lower level (−120 mV)
after 20 h. Generally, rNAD+/rNADH ratio was closely and
positively associated with the NAD+/NADH ratio. It was
CERðt Þ ¼ r F1 F2 F3 U
CO2 ðt Þ þ r CO2 ðt Þ þ r CO2 ðt Þ−r CO2 ðt Þ ð15Þ shown that in vivo rNAD+/rNADH ratio was at a lower level

r NADþ r F þ ðt Þ r F1
NADþ
ðt Þ þ r F2
NADþ
ðt Þ
ðt Þ ¼ FNAD ¼ F1
r NADH r NADH ðt Þ r NADH ðt Þ þ r NADH ðt Þ þ r F3
F2
NADH ð t Þ þ r F4 F5
NADH ðt Þ þ r NADH ðt Þ

r GLU ðt Þ þ 2OURðt Þ
¼ F3
2r GLU ðt Þ þ r F1 F2
CO2 ðt Þ þ r CO2 ðt Þ þ r CO2 ðt Þ þ r 7

r GLU ðt Þ þ 2OURðt Þ r GLU ðt Þ þ 2OURðt Þ

¼ ¼
2r GLU ðt Þ þ CERðt Þ þ r 3 þ r 7 2r GLU ðt Þ þ CERðt Þ þ 2r 3 −r 8

r GLU ðt Þ þ 2OURðt Þ
¼
3r GLU ðt Þ þ CERðt Þ−r GLU ðt Þ
ð16Þ
Cao et al. Bioresources and Bioprocessing (2015) 2:9 Page 5 of 11

(a) 90 (b) 100

Glutamate concentration-1)(g/L)
75 80

60
60
OD620

45
40
30
20
15

0 0
0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35
Time (h) Time (h)
(c) 2.0 (d) 100

1.6 50

1.2
ORP (mV) 0
RQ (-)

0.8
-50
0.4
-100
0.0
5 10 15 20 25 30 35 0 5 10 15 20 25 30 35
Time (h) Time (h)
Figure 2 Comparison of fermentation parameters for normal and abnormal batches. Black square and solid line, normal batch; white
square and dotted line, abnormal batch without sorbitol or glycerol; arrow, glucose fed. a: Time courses of cell concentration (OD620) in different
operation conditions; b: Time courses of glutamate concentration in different operation conditions; c: Time courses of RQ in different operation
conditions; d: Time courses of ORP in different operation conditions.

and NADH was excessive in abnormal fermentation after abnormal fermentation was due to the change of the
20 h, which was verified by the lower rNAD+/rNADH ratio characteristics of strain which led to the following re-
calculated in Table 1. Therefore, the improper rNAD+/rNADH sults: (1) glutamate production ability decreased and (2)
ratio after glucose feeding might be the reason for fermentation environment changed after glucose was fed
the non-accumulation of glutamate in the abnormal and the strain failed to adapt to the change, resulting in
batch [17]. intracellular abnormal metabolism and stoppage of
The abnormal performance appeared after glucose was glutamate synthesis. It has been reported that some
fed, and it was not contaminated. It was speculated that osmoregulators (such as trehalose or betaine) are either

Table 1 rNAD+/rNADH ratio at different instants under different operation conditions

Time (h) Fermentation batches
Batch #1 Batch #2 Batch #3 Batch #4 Batch #5 Batch #6
14 1.11 0.85 1.10 0.78 1.03 0.90
16 1.11 0.92 1.13 0.85 0.94 0.94
18 1.02 0.83 1.15 0.83 0.98 0.90
20 0.94 0.66 1.10 0.89 0.93 0.88
22 0.93 0.58 1.08 0.84 0.97 0.87
24 0.89 0.56 1.15 0.95 0.89 0.91
26 0.93 0.59 1.09 0.96 0.94 0.86
28 0.72 0.64 0.95 0.82 0.76 0.82
Batch #1, normal batch (control); batch #2, abnormal batch without sorbitol or glycerol; batch #3, sorbitol co-fed with glucose; batch #4, 15 g/L sorbitol added in
the initial medium; batch #5, glycerol co-fed with glucose; batch #6, 10 g/L glycerol added in the initial medium.
Cao et al. Bioresources and Bioprocessing (2015) 2:9 Page 6 of 11

produced by the microorganisms or taken up from the change much despite the sorbitol supplement methods
medium in lysine production by C. glutamicum in re- adopted. Glutamate production did not cease and final
sponse to a hyperosmotic shock [18,19]. They can pro- glutamate concentration reached 73 and 77 g/L at 34 h,
tect cells against environmental shock/stress. Sorbitol respectively, when co-feeding sorbitol with glucose or
has the same effect. When glutamate accumulation ceased adding sorbitol in the initial medium. No difference in
and the apparent fermentation parameters (OUR, CER, RQ before 20 h in the batches with/without sorbitol was
etc.) declined after glucose feeding for a period of 2 h, observed. RQ decreased continuously after 20 h in the
then ‘abnormal’ fermentation status was concluded. presence of sorbitol (RQ was about 0.4 at 34 h). Lower
Twelve and 2 g/L sorbitol were added at 26 and 32 h, RQ was favorable for glutamate accumulation and indi-
respectively; glutamate concentration increased grad- cated that less glucose proceeded beyond the α-KG node
ually and reached 72 g/L at 36 h with a 2-h fermenta- in the TCA cycle [20]. In these cases, less NADH was
tion period extend, as shown in Figure 3. accumulated in vivo and it was desirable for maintaining
Therefore, the abnormal fermentation was due to the de- cellular activities [21]. Less NADH accumulation implied
crease of resistance ability in response to the environmental that r(F)NADH is less than r(C)NADH, leading to a higher
alterations. In addition, the permeability of cell membrane rNAD+/rNADH ratio as well as a higher ORP levels. Under
increased to secrete glutamate extensively in the production the condition of co-feeding sorbitol with glucose, ORP
phase, and the glucose addition easily brought about shock was maintained at a normal level (−75 to −85 mV) after
or stress in the living environment. The carbon flux distri- feeding the mixed carbon sources. When 15 g/L sorbitol
bution in vivo was adjusted, and consequently, the metabol- was added in the initial medium, ORP always decreased in
ism of NAD+ and NADH was changed. On the other hand, the production phase, but it was much higher than that in
PDH and ICDH required NAD+ as the coenzyme and less the abnormal batch without sorbitol. rNAD+/rNADH ratio
NAD+ amount restricted the enzymes' catalytic actions. As was more than 0.8 after 20 h when co-feeding sorbitol
a result, the metabolic flux was redistributed and a series of with glucose or adding sorbitol in the initial medium, as
abnormal effects arose. shown in Table 1. The deterioration of the fermentation
Cells might be more tolerant to the environmental performance was reversed in the presence of sorbitol. This
change if some osmoregulators, such as trehalose or might be because the resistance to environmental change
betaine, were added before or at the same time when the was enhanced and NADH consumption was returned to
environmental shock/stress occurred. However, trehalose normal. The rNAD+/rNADH ratio was returned to normal
and betaine are expensive. Sorbitol and glycerol were level in the presence of sorbitol, and higher ORP values
cheaper, and they were also efficient environmental shock/ supported this fact indirectly.
stress protective reagents. Hence, fermentation perform-
ance when co-feeding sorbitol/glycerol with glucose or Fermentation performance in presence of glycerol
adding sorbitol/glycerol in the initial medium was studied. The fermentation performance when co-feeding glycerol
with glucose or adding glycerol in the initial medium is
Fermentation performance in presence of sorbitol shown in Figure 5. Similar fermentation curves and per-
The fermentation performance in presence of sorbitol is formance as those in the presence of sorbitol were ob-
shown in Figure 4. The cell growth patterns did not tained. The final glutamate concentration also reached

(a) (b)
80 100
Glutamate concentration) (g/L)

80
60

60
3
OD620

40
2
40 1

20
20

0 0
0 10 20 30 40 0 10 20 30 40
Time (h) Time (h)
Figure 3 Changing patterns of cell and glutamate concentrations under the condition of supplementing sorbitol when glutamate
accumulation ceased. Black square, normal batch; white square, abnormal batch with addition of sorbitol; arrow 1, glucose fed; arrow 2 and
arrow 3, adding 12 and 2 g/L sorbitol, respectively. a: Time courses of cell concentration (OD620) in different operation conditions; b: Time courses
of glutamate concentration in different operation conditions.
Cao et al. Bioresources and Bioprocessing (2015) 2:9 Page 7 of 11

(a) 100 (b) 100

Glutamate concentration-1)(g/L)
80 80

60 60
OD620

40 40

20 20

0 0
0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35
(c) 2.0 Time (h) (d) 100 Time (h)

1.6 50

1.2
ORP (mV) 0
RQ (-)

0.8
-50
0.4
-100
0.0
5 10 15 20 25 30 35 0 5 10 15 20 25 30 35
Time (h) Time (h)
Figure 4 Comparison of fermentation parameters with/without sorbitol. White square and dotted line, abnormal batch without sorbitol;
black square and dashed line, sorbitol co-fed with glucose; black circle and solid line, 15 g/L sorbitol added in the initial medium; arrow,
glucose fed. a: Time courses of cell concentration (OD620) in different operation conditions; b: Time courses of glutamate concentration in
different operation conditions; c: Time courses of RQ in different operation conditions; d: Time courses of ORP in different operation conditions.

normal levels at 34 h (72 and 76 g/L, respectively) when Investigation of the role of sorbitol and glycerol during
co-feeding glycerol with glucose or adding glycerol in glutamate fermentation
the initial medium. The improvements in glutamate produc- The concentrations of sorbitol and glycerol were assayed,
tion were also closely associated with higher rNAD+/rNADH and results are shown in Figure 6. The results indicated that
ratio (more than 0.8) as shown in Table 1, which was sorbitol and glycerol were hardly utilized by C. glutamicum
also reflected by higher ORP values (Figure 5) and when co-feeding them with glucose or adding them in the
lower RQ (0.6 to 0.7) in the late production phase. initial medium. When sorbitol or glycerol was co-fed with
From the results above, it could be concluded that ab- glucose, sorbitol and glycerol did not reduce after feeding;
normal glutamate fermentations could be restored to instead, they were gradually accumulated in the broth.
normal by supplementing the media with sorbitol or gly- While only a small portion of sorbitol or glycerol could
cerol, especially when sorbitol or glycerol was added in be consumed when they were added in the initial
the initial medium. It has been reported that sorbitol and medium. In summary, sorbitol and glycerol were not
glycerol could be assimilated by yeast and Escherichia coli assimilated by C. glutamicum but functioned as pro-
to increase the targeted product yield by effectively pro- tectants to improve the tolerance of the strain in re-
viding the required energy [22,23]. Furthermore, both sponse to the disturbance of the living environment.
sorbitol and glycerol were used as effective protective The glutamate biosynthesis pathway is composed of
agents of cell viability and enzymes due to their hygro- many enzymatic reactions in which citrate synthase
scopicity, freezing tolerance, and oxidation resistance. The (CS), PDH, and ICDH are the key enzymes directing car-
major role that sorbitol and glycerol played in the restor- bon flux towards TCA cycle (Figure 1a). Their activities
ation of abnormal glutamate fermentation was analyzed are repressed by excessive NADH. Lower rNAD+/rNADH
subsequently. ratio indicates that NADH was more than NAD+ in the
Cao et al. Bioresources and Bioprocessing (2015) 2:9 Page 8 of 11

(a) 90 (b) 100

Glutamate concentration-1)(g/L)
75 80
60
60
OD620

45
40
30

15 20

0 0
0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35
(c) 2.0 Time (h) (d) 100 Time (h)

1.5 50

ORP (mV) 0
RQ (-)

1.0

-50
0.5

-100
0.0
5 10 15 20 25 30 35 0 5 10 15 20 25 30 35
Time (h) Time (h)
Figure 5 Comparison of fermentation parameters with/without glycerol. White square and dotted line, abnormal batch without glycerol;
black square and dashed line, glycerol co-fed with glucose; black square and solid line, 10 g/L glycerol added in the initial medium; arrow, glucose fed.
a: Time courses of cell concentration (OD620) in different operation conditions; b: Time courses of glutamate concentration in different operation
conditions; c: Time courses of RQ in different operation conditions; d: Time courses of ORP in different operation conditions.

cytoplasm. PDH and ICDH activities could be limited by PDH, ICDH, and CcO before and after glucose addition
the insufficiency of NAD+. CcO is the key enzyme cata- (also mixed substrates) under different operation condi-
lyzing the transformation of proton (H+) from NADH to tions were analyzed. The relative activity of each enzyme
O2 through the respiratory chain [24]. During this period, before feeding glucose (also mixed substrates, 18 h) was
NADH was consumed and NAD+ was regenerated. The defined as 1. The REA after feeding (26 h) are shown in
activity of CS was difficult to measure, so the activities of Table 2. In the abnormal fermentation without sorbitol or

(a) 20
(b) 20
concentration (g/L)

concentration (g/L)

15 15
Sorbitol/Glycerol

Sorbitol/Glycerol

10 10

5 5

0 0
15 20 25 30 35 0 5 10 15 20 25 30 35
Time (h) Time (h)
Figure 6 Time courses of sorbitol and glycerol concentrations under different supplementing conditions. (a) Sorbitol or glycerol co-fed
with glucose; (b) sorbitol or glycerol added in the initial medium. Black square, sorbitol; black circle, glycerol.
Cao et al. Bioresources and Bioprocessing (2015) 2:9 Page 9 of 11

Table 2 Relative enzymatic activities of PDH, ICDH, and CcO after substrate(s) feeding (26 h) in different batches
Key enzyme Fermentation batches
Batch #1 Batch #2 Batch #3 Batch #4 Batch #5 Batch #6
PDH 0.84 ± 0.057 0.58 ± 0.043 0.72 ± 0.052 0.72 ± 0.036 0.76 ± 0.078 0.72 ± 0.061
ICDH 1.18 ± 0.051 0.72 ± 0.039 0.97 ± 0.061 0.92 ± 0.032 1.24 ± 0.076 1.27 ± 0.092
CcO 0.50 ± 0.063 0.00 1.50 ± 0.089 0.79 ± 0.043 0.75 ± 0.057 0.25 ± 0.04
Batch #1, normal batch (control); batch #2, abnormal batch without sorbitol or glycerol; batch #3, sorbitol co-fed with glucose; batch #4, 15 g/L sorbitol added in
the initial medium; batch #5, glycerol co-fed with glucose; batch #6, 10 g/L glycerol added in the initial medium.

glycerol, the activities of PDH and ICDH decreased by 42 pathway of glutamate synthesis. Consequently, glutamate
and 28%, respectively, and CcO was inactive after glucose accumulated continuously after glucose feeding and a
feeding. In the presences of sorbitol or glycerol, the inacti- failed fermentation could be avoided.
vation of these enzymes was relieved. The activity of PDH Sorbitol and glycerol served as shield materials during glu-
decreased by 20 to 30%, and ICDH activity was not sig- tamate fermentation. The activity of key enzymes could be
nificantly affected. The activity of CcO was almost properly maintained when supplementing sorbitol or gly-
maintained at a higher level. It was concluded that the cerol. The rNAD+/rNADH ratio was increased, and ORP was
repression of the key enzymes directing glucose into maintained around the normal range. The stability of glu-
glutamate synthesis was relieved by addition of sorbitol tamate fermentation was improved efficiently by adding
or glycerol. Accompanied by the recovery of NAD+ regen- sorbitol or glycerol, and the improvement was more obvious
eration, the metabolic flux was shifted into the normal when sorbitol/glycerol was added in the initial medium.

(a) 90 (b) 100

Glutamate concentration-1 (g/L)

75 80
60
60
OD620

45
40
30

15 20

0 0
0 10 20 30 40 0 10 20 30 40
(c) Time (h) (d) 100 Time (h)
2.0

1.5 50
ORP (mV)

0
RQ (-)

1.0

-50
0.5

-100
0.0
0 10 20 30 40 0 10 20 30 40
Time (h) Time (h)
Figure 7 Glutamate fermentation performance when less amount of sorbitol or glycerol was added in the initial medium. White square
and dotted line, abnormal batch without sorbitol or glycerol; black square and dashed line, 5 g/L sorbitol added in the initial medium; black circle
and solid line, 4 g/L glycerol added in the initial medium; arrow, glucose fed. a: Time courses of cell concentration (OD620) in different operation
conditions; b: Time courses of glutamate concentration in different operation conditions; c: Time courses of RQ in different operation conditions;
d: Time courses of ORP in different operation conditions.
Cao et al. Bioresources and Bioprocessing (2015) 2:9 Page 10 of 11

Feasibility analysis in industry Authors' contributions

A likely failed fermentation could be restored to normal YC carried out the experiments, performed the statistical analysis, and
drafted the manuscript. Z-NH was involved in performing the experiments.
when co-feeding sorbitol or glycerol with glucose or ME helped carry out the experiments and revised the manuscript. Z-PS
adding them in the initial medium. However, glutamate conceived the idea, participated in its design and coordination, and
is a low value-added product, and the supplementing helped in drafting of the manuscript. All authors read and approved the
final manuscript.
amount of sorbitol or glycerol should be minimized to
save the raw-material cost in industry. Therefore, the Acknowledgements
cell growth and glutamate production were analyzed The authors thank the financial sponsors from the National High-Tech Program
with less sorbitol or glycerol (4 to 5 g/L) addition in the (#2006AA020301) and Major State Basic Research Development Program
(#2007CB714303) of China.
initial medium, as shown in Figure 7. In these cases, cell
growth was not affected and glutamate synthesis did not Author details
1
stop after feeding glucose and glutamate concentration National University of Singapore (Suzhou) Research Institute, 377 Linquan
Street, Suzhou, Jiangsu Province, China. 2School of Biotechnology, Jiangnan
ended at 74 g/L at 36 h. It was verified again that sorb- University, 1800 Lihu Road, Wuxi, Jiangsu Province, China. 3Department of
itol and glycerol functioned mainly as protectants, and Food Processing Technology, Harare Institute of Technology, 1505 Ganges
their protective effect strengthened with increasing con- Road, P.O. Box BE 277, Belvedere, Harare, Zimbabwe.

centration of shield materials [8]. Received: 26 August 2014 Accepted: 22 December 2014

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