Bioprocess Biosyst Eng (2017) 40:891–899
DOI 10.1007/s00449-017-1753-7
RESEARCH PAPER
High-efficient production of citric acid by Aspergillus niger
from high concentration of substrate based on the staged-addition
glucoamylase strategy
Baoshi Wang1,2,3 · Hua Li1 · Linghuan Zhu1,2 · Fengling Tan3 · Youran Li1,2 ·
Liang Zhang1,2 · Zhongyang Ding1,2 · Guiyang Shi1,2 
Received: 3 November 2016 / Accepted: 14 February 2017 / Published online: 7 April 2017
© Springer-Verlag Berlin Heidelberg 2017
Abstract  Citric acid (CA), an important platform-com-
pound, has attracted much attention because of its broad
applications and huge market demand. To solve high resid-
ual sugar at the fermentation end, we put forwarded strat-
egy of pre-saccharification and then fermentation. Results
showed that the residual total sugar decreased by 10.4%
and the productivity increased by 4.0% and initially high
glucose inhibited cell growth. Furthermore, commercial
glucoamylase with high low-pH stability was proposed to
staged-add in the fermentation process, which timely com-
pensated enzyme loss, ensuring the glucose supply rate.
The fermentation productivity was evidently enhanced by
13.3% with residual total sugar decreasing by 31.3%, sim-
plifying the subsequent product separation and extraction
process. Our results confirmed that staged-addition glucoa-
mylase strategy was feasible to effective production of CA.
Keywords  Staged-addition strategy · Glucoamylase ·
Citric acid · Simultaneous saccharification and
fermentation · Aspergillus niger
* Guiyang Shi
gyshi@jiangnan.edu.cn
1
National Engineering Laboratory for Cereal Fermentation
Technology, Wuxi 214122, People’s Republic of China
2
School of Biotechology, Jiangnan University, Wuxi 214122,
People’s Republic of China
3
Jiangsu Guoxin Union Energy Co., Ltd., Wuxi 214203,
People’s Republic of China
Introduction
Citric acid (2-hydroxy-1, 2, 3-propanetricarboxylic acid,
CA) is widely used in food, pharmaceutical, and other
industries due to its unique attributes [1–3]. Particularly,
as the most mature platform-compound, CA has become
the world’s largest organic acid [4]. Currently, the global
demand is continuously increasing at a high annual rate
of 5%, along with new applications in the advanced fields
such as nanomedicine, drug delivery, and tissue engineer-
ing [5–7]. Consequently, the efficient biosynthesis of CA is
very important to cover the ever-increasing demand.
CA is a naturally occurring organic acid that can be pro-
duced by chemical synthesis or biological synthesis [1].
Biosynthesis method has the advantages of low energy con-
sumption and renewable resources use [8]. Understandably,
more than 99% of CA is produced by microbial fermenta-
tion [9]. Filamentous fungus A. niger is a good microbial
cell factory for CA production due to the superiorities in
ease of handling, broad substrate, and high yields [10]. In
addition, A. niger can secrete multiple hydrolytic enzymes
such as α-amylase and glucoamylase into the broth during
fermentation. In addition, this is significantly important to
CA production directly from starchy materials [11].
As for the CA production, carbon sources could be used
(Table  1). Using the refined sugars can achieve higher
yield, but it is costly to be commercially used [12]. Raw
starchy material is more economical and competitive in the
industrial production [13]. However, the starchy material
cannot be used as the direct carbon source, and it requires
complex pretreatment process such as gelatinization and
liquefaction, followed by saccharification process to the
fermentable sugars [11, 14]. To reduce the production cost,
saccharification process and fermentation process could be
integrated into one step as simultaneous saccharification
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892 Bioprocess Biosyst Eng (2017) 40:891–899

Table 1  Citric acid production with different carbon sources

Strains Sugar source Titer (g/L) Productivity Fermentation system (scale) References
(g/L/h)

A. niger Corn hyrolysate 185.7 2.58 Fermentor (30 L) Wang et al. [11]
A. niger Cassava hyrolysate 141.5 1.97 Bioreactor (5 L) Xu et al. [22]
A. niger Corn hyrolysate 151.7 1.58 Fermentor (30 L) Wang et al. [23]
A. niger Corn hydrolysate 187.5 3.13 Bioreactor (50 L) Hu et al. [24]
A. niger Black-strap molasses 86.1 0.51 Conical flask (300 mL) Khurshid et al. [25]
A. niger Ram-horn hydrolysate 94.1 0.78 Fermenter (2 L) Kurbanoglu et al. [26]
Y. lipolytica Glucose 101.0 0.42 Fermenter (10 L) Tan et al. [27]
Y. lipolytica Waste cooking-oil 31.7 0.09 Fermenter (10 L) Liu et al. [28]
Y. lipolytica Sucrose 140 0.09 Bioreactor Förster et al. [29]
(2 L)

and fermentation (SSF). The SSF process was advantageous
in solution of high-sugar inhibition and has been developed
and successfully applied in ethanol [15], organic acid such
as fumaric acid [16], itaconic acid [13], and lactic acid [14]
production. As for the CA industrial production from the
starchy material, SSF mode was extensively applied, since
the producing strain A. niger itself could secrete abundant
glucoamylase [17–21]. However, there existed a big prob-
lem that pH sharp-decrease (below 2.00) with the CA accu-
mulation, significantly damaging the glucoamylase activity.
The resulting loss of glucoamylase activity influenced the
glucose supply and strongly decreased the CA production
rate. Moreover, it normally resulted in higher total sugar
remaining in the broth at the fermentation end. These prob-
lems not only reduce the conversion ratio of substrate to
CA, but also interfere with the subsequent separation and
purification steps [14]. Higher residual sugar could eas-
ily cause the RCS (readily carbonizable substances) of the
products beyond the standard. Complex devices such as
membrane separation and column chromatography were
required to invest to remove the higher sugar, which further
raised the production costs.
To decrease the residual sugar concentration, Huang
et  al. overexpressed the glucoamylase gene in A. terreus
along with the itaconic acid production enhanced directly
from the liquefied corn starch [13]. Similarly, CA produc-
tion was improved through overexpression the glucoa-
mylase gene in A. niger [11]. Although genetic engineer-
ing have made some improvements, the instability of the
recombinant strains and the concerns over food safety must
be considered, especially when CA is applied in food addi-
tives and healthcare fields. Recently, staged-control strate-
gies such as pH control, speed control, and dissolved oxy-
gen have been successfully developed and applied in the
amino acid and organic acid production [30–33]. In addi-
tion, it could efficiently enhance the productivity due to
better adapting to the characteristics of strain growth and
product synthesis. Collectively, these developments of
enhancing the glucoamylase activity and staged-control
strategies provided great inspirations to cope with the high
residual sugars in CA production.
In this study, direct production of CA from liquefied
starch at high concentration substrate was achieved based
on the staged-addition strategy. Commercial glucoamylase
with high low-pH stability was stage-added in the fermen-
tation process and timely compensated the enzyme loss,
ensuring the glucose supply.
Materials and methods
Strain
Aspergillus niger used in this study was stored in Jiangsu
Guoxin Union Energy Co., Ltd, which was an industrial
strain for CA production. Fresh spores were cultured at
35 °C for 7 days in the eggplant-flask. The spore suspension
was prepared by adding distilled water with Tween-80 (2%,
v/v) and then was used for inoculation.
Medium preparation and culture conditions
Corn flour liquefaction slurry (DE value, 26.2%) and lique-
faction filtrate (DE value, 24.6%) were kindly provided by
Jiangsu Guoxin Union Energy Co., Ltd. (Yixing, China).
Seed medium contained the liquefaction slurry (initial
total sugar approximately 80 g/L) and (­ NH4)2 ­SO4 addition
2 g/L.
Spore suspension (1 mL containing 4.0 × 107 spores)
was inoculated in a 500 mL conical flask with 100 mL
seed medium and was incubated on rotary orbital shaker
at 320 rpm and 35 °C for 28 h. Fermentation medium con-
sisted of the liquefaction slurry and the liquefaction filtrate
and tap water with the initial total sugar approximately

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Bioprocess Biosyst Eng (2017) 40:891–899 893
175  g/L. Shake-flask fermentations were carried out in a
250 mL flask with 40 mL fermentation medium, and inoc-
ulated with 4 mL seed-culture and then cultured at 35 °C
with shaking at 320 rpm for 84 h. In addition, a 24 L fer-
menter with 16 L working volume was performed at 35 °C
with dissolved oxygen (DO) at 50%.
Pre‑saccharification using the commercial glucoamylase
for CA production
A 24  L-fermenter with 14.6  L fermentation medium was
sterilized at 115 °C for 20  min. After sterilization, com-
mercial glucoamylase (provided by Novozymes China Co.
Ltd.) with the volume 10 mL (total activity of the enzyme
2.8 × 105 U) was added when the temperature decreased to
60 °C and maintained for 2  h. Mature seed broth (1.6  L)
was inoculated to the fermenter to start the CA fermenta-
tion when the temperature decreased to 35 °C. Fermenta-
tions were considered to be over when the residual glucose
was below 0.5  g/L. Agitation was provided by three six-
bladed disk turbines. The aeration rate was 2.0 vvm and the
DO coupled with agitation speed was at 50%.
Analysis of the enzymatic properties of A. niger
glucoamylase and commercial glucoamylase
Glucoamylase was preliminarily purified by a modified
method of Riaz et al. [34]. The supernatant of the fermenta-
tion broth (cultivated at 24 h) was collected by centrifuga-
tion at 10,000×g, 4 °C for 20  min. Solid ammonium sul-
phate was added gradually to the supernatant and diluent
solutions of commercial glucoamylase to 60% saturation at
0 °C and left overnight at 4 °C. Centrifuged under the same
conditions, the supernatant was obtained. Solid ammonium
sulphate was further added to the supernatant to achieve
the final 90% saturation at 0 °C and left overnight at 4 °C.
Repeated the above centrifugation process, the precipita-
tion contained glucoamylase was obtained. Then, the pre-
cipitation was dissolved in distilled water and dialyzed over
night against several changes of distilled water at 4 °C to
remove salt and other ingredients.
Glucoamylase activity was determined by the modi-
fied method of Iqbal et  al [35]. Enzymatic reaction con-
tained 2% (w/v) soluble starch (5 mL) as the substrate
and the enzyme (100 μL) in 50  mM Na-acetate buffer
(pH 4.60) with a total volume of 6 mL and was incubated
at 40 °C for 20 min. The reaction was stopped by immer-
sion in boiling water for 5 min and then examined using
DNS method [36]. Glucoamylase activity was quantified
by measuring the amount of reducing sugar using DNS
method. One unit of enzymatic activity was tentatively
defined as equal to the reducing power per hour at 40 °C
and pH 4.6. Effects of pH on glucoamylase activity and
stability were investigated. Enzyme activity was deter-
mined at various pH values at 40 °C. The stability was
assayed after the purified enzymes was incubated for dif-
ferent times in buffers of pH values of 1.80, 1.90, 2.00,
and 2.10.
Evaluation of glucoamylase addition strategy on CA
production
Optimization experiments of enzyme addition strategy
were all performed in shake flask at 320  rpm and 35 °C
for 84  h. Glucoamylase addition level was based on the
difference values between the remaining total sugar and
glucose concentration. Glucoamylase addition condi-
tions including addition level (100, 300, 600, and 1000
U/g; addition pH 2.10; addition at one time), addition pH
(2.50, 2.10, 1.90, and 1.80; addition level 600 U/g; addi-
tion at one time) and addition strategy (addition at one
time, addition at intervals of 3, 6, and 9 h; addition level
600 U/g; addition pH 2.10) was investigated.
CA fermentation with glucoamylase staged‑addition
strategy
Based on the above-optimized conditions (enzyme addi-
tion level 600 U/g, addition pH value 2.10, addition
strategy at intervals of 6 h), CA fermentations were per-
formed in a 24  L fermenter with 16  L working volume
and cultured under the same operational conditions as
mentioned above.
Analytical methods
To analyze the residual total sugar and CA yield, the fer-
mentation culture was centrifuged at 10,000×g and 4 °C
for 10 min, and the clear supernatant was filtered through
a 0.22 μm syringe filter for analysis. Glucose concentra-
tion was determined by a biosensor (SBA-40B, Shan-
dong Academy of Sciences, China) [37]. Residual total
sugar in the broth was determined by DNS method [36]
after the samples were subjected to acid hydrolysis on
the heating panel (Stainless Steel Electric Heating Board
C-MAG HP7, IKA) at 500 °C boiling for 5 min. CA was
determined by high-performance liquid chromatography
using SH1011 column (Agilent, USA) and subsequently
detected at 210  nm with a UV detector (UVD 170U,
DIONEX Co., Ltd.) with mobile phase 0.01 M ­H2SO4 at
50 °C.
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Results and discussion
Pre‑saccharification and fermentation strategy for CA
production
Based on the saccharifying ability of the A. niger itself, CA
fermentation was industrially performed in the SSF mode.
Unfortunately, pH value sharply declined in the broths with
CA accumulation where the glucoamylase activity was
greatly destroyed. As a result, massive total sugar (approxi-
mately 20  g/L) remained in the broth at the fermentation
end.
To decrease the residual sugar concentration and
enhance the fermentable sugar in the medium, we pro-
posed pre-saccharification and fermentation (PSF) strat-
egy. Glucoamylase was added at 60 °C after sterilization
and maintained for 2  h and then fermentation was con-
ducted. As expected, the glucose concentration in the initial
medium was significantly increased to 102.5 g/L (the con-
trol, 16.5  g/L, Fig.  1c). CA production attained 170.6  g/L
after 59 h in comparison with the control (169.4 g/L, 61 h)
(Fig. 1 a). Surprisingly, the CA production rate in the first
24  h was a little lower than the traditional fermentation
mode (Fig.  1f). These phenomenon might result from the
higher glucose concentration at the initial stage due to the
imbalance in osmotic pressure. This inhibition effect was
gradually relieved with the glucose concentration reduc-
tion and the strain adaptability. As shown in Fig.  1f, CA
Fig. 1  Citric acid fermentations by pre-saccharification and fermentation strategy in a 24 L-fermenter. a citric acid production, b residual total
sugar, c residual glucose, d pH, e glucoamylase activity, and f citric acid production rate. Data are means ± SD (n = 3)
13
Bioprocess Biosyst Eng (2017) 40:891–899
production rate even exceeded the control due to more suf-
ficient available sugar after 24 h.
As known to us, glucoamylase activity plays a vital role
in the targeted products accumulation in the SSF mode.
Surprisingly, glucoamylase activities in the traditional pro-
cess rapidly increased in the initial stage and then signifi-
cantly declined with the pH sharp decrease (Fig. 1 d, e). In
contrast, enzyme activities in the PSF process were a lit-
tle higher since the added glucoamylase partially remain-
ing in the broth. Residual total sugar decreased by 10.4%
compared with the control. Nevertheless, there is still a
great loss of glucoamylase activity in the later period of
the fermentation (Fig. 1e), resulting in 17.2 g/L total sugar
remained.
Taken together, PSF mode partly enhanced the CA syn-
thesis rate, while high glucose at the initial stage inhibited
the cell growth. Simultaneously, there is still a great loss
of the glucoamylase activity in later fermentation period,
along with relatively higher residual sugar at the fermenta-
tion end. To further improve the CA productivity, compen-
sation of the enzyme activity loss in SSF process was quite
necessary.
Enzymatic properties of A. niger glucoamylase
and commercial glucoamylase in the low pH values
Sufficient glucoamylase activity played an impor-
tant role in providing adequate glucose directly from
Bioprocess Biosyst Eng (2017) 40:891–899 895
starchy materials, especially in the low-pH environment.
Although our proposed PSF strategy partly improved
the CA production rate, glucoamylase activity was still
reduced with the pH decreasing at later stage of the fer-
mentations. Therefore, selection of a better pH-tolerance
glucoamylase added in the fermentation process was
necessary to compensate the enzyme activity loss.
Enzymatic properties of the purified commercial
glucoamylase and A. niger glucoamylase at different
pH values were systematically investigated as shown in
Fig.  2. Surprisingly, optimum pH for the two enzymes
was both 4.60 while commercial enzyme had a rela-
tively broader pH range, especially during the low pH
conditions (Fig. 2a, b). Simultaneously, stabilities in the
low pH conditions with different incubation time were
investigated. To simulate the low-pH environment in
the CA fermentations, pH values were set as 2.10, 2.00,
1.90, and 1.80, respectively. On the whole, commercial
glucoamylase was more stable than that of A. niger, as
shown in Fig. 2c and d. In addition, it could even remain
more than 70% activity after incubated at pH 1.80 for
10  h, while A. niger glucoamylase activity decreased
below 50% (Fig. 2c, d). With this unique attributes, com-
mercial glucoamylase was suitable to be used in the CA
fermentation process, which could timely compensate
the enzyme activity loss to ensure the glucose supply.
Fig. 2  Effects of pH on the
activity and stability of Asper-
gillus niger glucoamylase (a, c),
commercial glucoamylase (b,
d). Data are means ± SD (n = 3)
Strategies of glucoamylase addition in the fermentation
process for CA production
To ensure adequate glucoamylase, activity retained in the
fermenter at all times is crucial for the CA accumulation in
the SSF mode. Nevertheless, continuous decline in pH with
CA accumulation caused a great loss in glucoamylase activ-
ity, restricting the glucose supply. To strengthen the fer-
mentation process, glucoamylase addition conditions such
as addition level, addition Ph, and addition strategy were
further investigated in the shake flask. To identify the influ-
ence of addition level on fermentation, addition levels (100,
300, 600, and 1000; U/g) under the conditions of addition
at one time from pH 2.10 were analyzed in a shaken-flask
experiment. With the increase of addition level from 100 to
600 U/g, CA production increased from 159.2 to 162.2 g/L.
However, it increased only 0.3 g/L (162.5 g/L) when con-
tinuously enhanced to 1000 U/g (Fig.  3a). In correspond,
the remaining total sugar decreased from 26.5 to 22.8 g/L
with a decrease of 14.0% and much more sugar was used to
synthesis CA (Fig. 3b). These findings were perfectly per-
sistent with the previous reports that higher glucoamylase
activity showed enhanced fumaric acid production [38]. In
the following experiment, we selected the addition level
600 U/g to be investigated.
In the foregoing PSF process, the initially high glucose
concentration inhibited the cell growth and CA produc-
tion rate. Thus, selection of appropriate time to add the
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Fig. 3  Influence of glucoamylase addition level (a, b addition pH
2.10 at one time; addition level based on the difference value between
the remaining total sugar and glucose concentration), addition pH(c,
d addition level 600 U/g; addition at one time), and addition strategy
enzyme was significantly important. To identify the influ-
ence of addition pH on fermentation, addition pH (2.50,
2.10, 1.90 and 1.80) under the conditions of addition level
600 U/g at one time were investigated (Fig. 3c, d). It was
clearly found that the CA production reached the high-
est (162. 5 g/L) at pH 2.10, along with the lowest residual
total sugar (22.5 g/L), compared to the control (158.1 g/L,
remaining total sugar 26.9 g/L). Actually, the CA produc-
tion rate and glucoamylase activity both attained the peak
at pH 2.10 (Fig.  1d–f). Thus, this timely glucoamylase
addition contributed to sufficient glucose release, rein-
forcing the SSF process. However, higher pH (2.50) could
generate more glucose, while CA production (161. 5  g/L)
was 1.0 g/L lower than that at pH 2.10, which might result
from the high sugar inhibition effect. Improvement of CA
titer was also lower when enzyme added at lower pH (1.90,
1.80) due to the restriction of glucose supply in the primary
stage. Consequently, glucoamylase addition at pH 2.10
could efficiently improve the CA production.
Throughout the CA fermentation process, the glucoa-
mylase activity presented a trend where the activity firstly
increased, and then decreased gradually owing to the pH
decline with the CA accumulation. To meet the glucoa-
mylase requirements at different stages, addition strate-
gies (a addition at one time, b–d staged addition at inter-
vals of 3, 6, and 9 h, respectively) under the conditions of
13
Bioprocess Biosyst Eng (2017) 40:891–899
(e, f addition level 600 U/g; addition pH 2.10) on the citric acid pro-
duction and residual total sugar (a represented the glucoamylase addi-
tion at one time, b–d represented the glucoamylase addition at inter-
vals of 3, 6, and 9 h, respectively). Data are means ± SD (n = 3)
addition level 600 U/g at pH 2.10 were investigated to fur-
ther improve the CA production, as shown in Fig.  3e and
f. As expected, staged-addition strategy distinctly enhanced
the CA production and reduced the remaining total sugar.
Interestingly, staged-addition strategy at intervals of 6  h
achieved the highest yield (166.3  g/L) compared with the
control (159.1  g/L), with an increase of 7.2  g/L. Corre-
spondingly, the residual total sugar significantly decreased
from 25.9 to 18.7 g/L, decreasing by 27.8%. These results
confirmed that staged-addition strategy could timely com-
pensate the glucoamylase loss, which better synchronized
with CA production.
Optimization of CA fermentation with glucoamylase
staged‑addition strategy in a 24 L fermenter
Based on the flask culture results, performances of CA pro-
duction with glucoamylase staged-addition strategy were
analyzed in a 24 L fermenter. As shown in Fig. 4d and e,
staged-addition strategy timely compensated the glucoam-
ylase activity loss due to the lower-pH caused by CA accu-
mulation. Surprisingly, CA production rate was evidently
accelerated (Fig.  4f) and the CA titer increased 3.8  g/L
compared with the control (169.4  g/L) in 6  h shorter fer-
mentation time (Fig.  4a). These results perfectly matched
with the previous results that higher glucoamyalse activity
Bioprocess Biosyst Eng (2017) 40:891–899 897

Fig. 4  Citric acid fermentation based on the glucoamylase staged-addition strategy in a 24 L-fermenter. a citric acid production, b residual total
sugar, c residual glucose, d pH, e glucoamylase activity, and f citric acid production rate. Data are means ± SD (n = 3)

showed improvement in the production of fumaric acid
[38], itaconic acid [13]. Moreover, the remaining total
sugar in the broth was dramatically decreased (by 31.3%)
from 19.2 to 13.2  g/L, remarkably enhancing the utiliza-
tion rate of starchy material. The reduction in sugar con-
centration was also favorable to the subsequent separation
and purification steps [39, 40]. Low residual sugar could
decrease the RCS of products, which could avoid the com-
plex devices investment for separation and purification.
Taken together, our established strategies could improve
the CA productivity and save the purification cost in the
viewpoint of practice and economy.
Different strategies including traditional fermentation
(the control), PSF strategy, and staged-addition strategy
were compared in Table 2. In comparison, strategy b and
strategy c presented advantages in improving the produc-
tivity. CA production increased 1.2 and 3.8  g/L with 2
and 6  h shorter fermentation time, respectively. In par-
ticular, the productivity of the s­trategyc increased from
2.78 to 3.15  g/L/h with an increase of 13.3%, distinctly
reducing the production cost. In addition, the residual
total sugar decreased by 31.3%, simplifying the subse-
quent product separation and purification process. Taken
together, these results confirmed that it is feasible and
efficient to produce CA by staged-addition glucoamylase
strategy.

Table 2  Fermentation Fermentation mode Citric acid produc- Residual total Fermentation Productivity (g/L/h)d
parameters in different tion (g/L) sugar (g/L) time (h)
fermentation strategies
Strategya 169.4 ± 5.4 19.2 ± 1.6 61 ± 3.0 2.78 ± 0.11
Strategyb 170.6 ± 6.2 17.2 ± 3.1 59 ± 2.6 2.89 ± 0.14
Strategyc 173.2 ± 4.1 13.2 ± 2.8 55 ± 2.0 3.15 ± 0.09

Data are means ± SD (n = 3)

a
 Control
b
 Pre-saccharification and fermentation
c
 Staged-addition strategy
d
 Concentration of citric acid (g/L)/fermentation time (h)

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Conclusions
12. John RP, G.S A, Nampoothiri KM, P Pandey A (2009) Direct
The present work successfully solved the problems of the
glucoamylase activity caused by the lower pH with CA
accumulation. Staged-addition strategy timely compensated
the glucoamylase activity loss and strengthened the SSF
process. Consequently, the remaining total sugar decreased
by 31.3% (from 19.2 to 13.2  g/L) and CA productivity
increased by 13.3% (from 2.78 to 3.15  g/L/h) compared
with the traditional fermentation process. This significant
decrease of the remaining total sugar was also beneficial to
the subsequent purification step and avoids complex device
investment. Collectively, these improvements will save the
production cost and facilitate the industrial application.
Acknowledgements  This research was financially supported by
the National High Technology Research and Development Program
of China (863 Program, 2015AA020302) and the Cooperation Pro-
ject of Jiangsu Province among Industries, Universities and Institutes
(BY2015019-13) and the Jiangsu Guoxin-Union Engergy Co. Ltd.,
China. We are thankful for their supports.
References
1. Singh Dhillon G, Kaur Brar S, Verma M, Tyagi RD (2011)
Recent advances in citric acid bio-production and recovery. Food
Bioprocess Technol 4:505–529
2. Yin X, Li J, Shin H-d, Du G, Liu L, Chen J (2015) Metabolic
engineering in the biotechnological production of organic acids
in the tricarboxylic acid cycle of microorganisms: Advances and
prospects. Biotech Adv 33:830–841
3. Papagianni M (2007) Advances in citric acid fermentation by
Aspergillus niger: Biochemical aspects, membrane transport and
modeling. Biotech Adv 25:244–263
4. Sauer M, Porro D, Mattanovich D, Branduardi P (2008) Micro-
bial production of organic acids: expanding the markets. Trends
Biotechnol 26:100–108
5. Yang J, Webb AR, Ameer GA (2004) Novel citric acid-based
biodegradable elastomers for tissue engineering. Adv Mater
16:511–516
6. Naeini AT, Adeli M, Vossoughi M (2010) Poly(citric acid)-
block-poly(ethylene glycol) copolymers—new biocompatible
hybrid materials for nanomedicine. Nanomed Nanotechnol Biol
Med 6:556–562
7. Guillermo A, Jian Y, Ryan H (2010) New biodegradable bio-
compatible citric acid nano polymers for cell culture growth
& implantation engineered by Northwestern University Sci-
entists. Nano patents and innovations. US Patent Application
20090325859
8. Carlos R., Soccol LPSV, Cristine Rodrigues, Pandey (2006) New
perspectives for citric acid production and application. Food
Technol Biotechnol 44:141–149
9. Thompson JC, He B (2006) Characterization of crude glycerol
from biodiesel production from multiple feedstocks. Appl Eng
Agric 22:261–265
10. Angumeenal AR, Venkappayya D (2013) An overview of citric
acid production. LWT Food Sci Technol 50:367–370
11. Wang L, Cao Z, Hou L, Yin L, Wang D, Gao Q, Wu Z,
Wang D (2016) The opposite roles of agdA and glaA on
13
Bioprocess Biosyst Eng (2017) 40:891–899
citric acid production in Aspergillus niger. Appl Microbiol
Biot 100:5791–5803
lactic acid fermentation: Focus on simultaneous saccharifica-
tion and lactic acid production. Biotech Adv 27:145–152
13. Huang X, Chen M, Lu X, Li Y, Li X, Li J-J (2014) Direct pro-
duction of itaconic acid from liquefied corn starch by geneti-
cally engineered Aspergillus terreus. Microb Cell Fact 13:1–10
14. Lv X, Yu B, Tian X, Chen Y, Wang Z, Zhuang Y, Wang Y
(2016) Effect of pH, glucoamylase, pullulanase and invertase
addition on the degradation of residual sugar in L-lactic acid
fermentation by Bacillus coagulans HL-5 with corn flour
hydrolysate. J Taiwan Inst Chem E 61:124–131
15. Szymanowska-Powałowska D, Lewandowicz G, Kubiak P,
Błaszczak W (2014) Stability of the process of simultaneous
saccharification and fermentation of corn flour. The effect of
structural changes of starch by stillage recycling and scaling
up of the process. Fuel 119:328–334
16. Saha BC, Nichols NN, Qureshi N, Kennedy GJ, Iten LB, Cotta
MA (2015) Pilot scale conversion of wheat straw to ethanol via
simultaneous saccharification and fermentation. Bioresource
Technol 175:17–22
17. Haq I-U, Ali S, Iqbal J (2003) Direct production of citric
acid from raw starch by Aspergillus niger. Process Biochem
38:921–924
18. Suzuki A, Sarangbin S, Kirimura K, Usami S (1996) Direct
production of citric acid from starch by a 2-deoxyglucose-
resistant mutant strain of Aspergillus niger. J Ferment Bioeng
81:320–323
19. Adeoyea AO, Lateefb A, Gueguim-Kana EB (2015) Optimiza-
tion of citric acid production using a mutant strain of Aspergil-
lus niger on cassava peel substrate. Biocatal Agric Biotechnol
4:568–574
20. Rao PR, Reddy MK (2013) Production of Citric Acid by
Aspergillus niger Using Oat Bran as Substrate. Int J Chem
Chem Eng 3:181–190
21. Mostafa YS, Alamri SA (2012) Optimization of date syrup for
enhancement of the production of citric acid using immobi-
lized cells of Aspergillus niger. Saudi J Biol Sci 19:241–246
22. Xu J, Chen Y-Q, Zhang H-J, Wang K, Tang L, Zhang J-H,
Chen X-S, Mao Z-G (2014) Optimization of the integrated
citric acid–methane fermentation process by air stripping and
glucoamylase addition. Bioprocess Biosyst Eng 38:411–420
23. Wang L, Zhang J, Cao Z, Wang Y, Gao Q, Zhang J, Wang D
(2015) Inhibition of oxidative phosphorylation for enhancing
citric acid production by Aspergillus niger. Microb Cell Fact
14:1–12
24. Hu W, Liu J, Chen JH, Wang SY, Lu D, Wu QH, Li WJ (2014)
A mutation of Aspergillus niger for hyper-production of cit-
ric acid from corn meal hydrolysate in a bioreactor. J Zhejiang
Univ Sci B 15:1006–1010
25. Khurshid S, Ali S, Ashraf H, Qadeer M, Rajoka MI (2001)
Mutation of Aspergillus niger for hyperproduction of citric
acid from black strap molasses. World J Microb Biot 17:35–37
26. Kurbanoglu EB (2004) Enhancement of citric acid production
with ram horn hydrolysate by Aspergillus niger. Bioresource
Technol 92:97–101
27. Tan M-J, Chen X, Wang Y-K, Liu G-L, Chi Z-M (2016)
Enhanced citric acid production by a yeast Yarrowia lipolytica
over-expressing a pyruvate carboxylase gene. Bioprocess Bio-
syst Eng 39:1–8
28. Liu XY, Lv JS, Xu JX, Zhang T, Deng YF, He JL (2015) Cit-
ric Acid Production in Yarrowia lipolytica SWJ-1b Yeast
When Grown on Waste Cooking Oil. Appl Biochem Biotech
175:2347–2356
Bioprocess Biosyst Eng (2017) 40:891–899 899
29. Förster A, Aurich A, Mauersberger S, Barth G (2007) Citric acid
production from sucrose using a recombinant strain of the yeast
Yarrowia lipolytica. Appl Microbiol Biot 75:1409–1417
30. Cheng L-K, Wang J, Xu Q-Y, Zhao C-G, Shen Z-Q, Xie X-X,
Chen N (2013) Strategy for pH control and pH feedback-con-
trolled substrate feeding for high-level production of l-trypto-
phan by Escherichia coli. World J Microb Biot 29:883–890
31. Sun J, Zhang L, Rao B, Han Y, Chu J, Zhu J, Shen Y, Wei D
(2012) Enhanced acetoin production by Serratia marcescens
H32 using statistical optimization and a two-stage agitation
speed control strategy. Biotechnol Bioproc E 17:598–605
32. Zhang J, Liu L, Li J, Du G, Chen J (2012) Enhanced glucosa-
mine production by Aspergillus sp. BCRC 31742 based on the
time-variant kinetics analysis of dissolved oxygen level. Biore-
sour Technol 111:507–511
33. Li X, Lin Y, Chang M, Jin Q, Wang X (2015) Efficient produc-
tion of arachidonic acid by Mortierella alpina through integrat-
ing fed-batch culture with a two-stage pH control strategy. Biore-
sour Technol 181:275–282
34. Riaz M, Rashid MH, Sawyer L, Akhtar S, Javed MR, Nadeem
H, Wear M (2012) Physiochemical properties and kinetics of
glucoamylase produced from deoxy-d-glucose resistant mutant
of Aspergillus niger for soluble starch hydrolysis. Food Chem
130:24–30
35. Iqbal Z, Rashid M, Jabbar A, Malana M, Khalid A, Rajoka
M (2003) Kinetics of enhanced thermostability of an extra-
cellular glucoamylase from Arachniotus sp. Biotechnol Lett
25:1667–1670
36. Ghose T (1987) Measurement of cellulase activities. Pure Appl
Chem 59:257–268
37. Xu J, Bao JW, Su XF, Zhang HJ, Zeng X, Tang L, Wang K,
Zhang JH, Chen XS, Mao ZG (2016) Effect of propionic acid
on citric acid fermentation in an integrated citric acid-methane
fermentation process. Bioprocess Biosyst Eng 39:391–400
38. Deng Y, Li S, Xu Q, Gao M, Huang H (2012) Production of
fumaric acid by simultaneous saccharification and fermentation
of starchy materials with 2-deoxyglucose-resistant mutant strains
of Rhizopus oryzae. Bioresource Technol 107:363–367
39. Anastassiadis S, Morgunov IG, Kamzolova SV, Finogenova TV
(2008) Citric acid production patent review. Recent Patent Bio-
technol 2:107–123
40. Joglekar H, Rahman I, Babu S, Kulkarni B, Joshi A (2006) Com-
parative assessment of downstream processing options for lactic
acid. Sep Purif Technol 52:1–17
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