Industrial Crops & Products 146 (2020) 112159

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Industrial Crops & Products

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The supplement of vitamin C facilitates L-lactic acid biosynthesis in T

Lactobacillus thermophilus A69 from sweet sorghum juice coupled with
soybean hydrolysate as feedstocks
Xuejiao Tiana,b, Wei Hua,b,*, Jihong Chena,b, Wei Zhangc, Wenjian Lia,b
a
Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou, PR China
b
University of Chinese Academy of Sciences, Beijing, PR China
c
Key Laboratory of Extreme Environmental Microbial Resources and Engineering, Northwest Institute of Eco-Environment and Resources, Chinese Academy of Sciences,
Lanzhou 730000, PR China

A R T I C LE I N FO A B S T R A C T

Keywords: Regulating oxidative damage is an effective way to enhance cell growth and metabolite accumulation in mi-
Vitamin C crobial strains. However, this strategy on lactic acid production has not been systematically reported yet. In
L-lactic acid present study, the effect of antioxidant vitamin C on cell growth and L-lactic acid production was investigated in
Sweet sorghum juice Lactobacillus thermophilus A69, and an addition of 75 mM vitamin C resulted in significant improvement in cell
Antioxidant capacity
growth and L-lactic acid accumulation. The total antioxidant capacity (T-AOC) and key enzyme activities in-
Fermentation efficiency
volving in glycolytic pathway (such as hexokinase, phosphofructokinase, pyruvate kinase, and lactate dehy-
drogenase) were also significantly enhanced by vitamin C addition. Finally, with the addition of optimal con-
centrations of vitamin C into sweet sorghum juice coupled with cheaper soybean hydrolysate as feedstocks, a
final L-lactic acid titer of 118.8 g/L with the productivity of 3.71 g/L/h was achieved by A69 strain. This study
provided an efficient and economical approach for enhancement of L-lactic acid fermentation efficiency using
sweet sorghum juice as a feedstock.

1. Introduction
Lactic acid is a versatile chemical used in food, leather tanning,
pharmaceuticals, and cosmetic industries (Datta and Henry, 2006; Gao
et al., 2011; Kong et al., 2019; Martinez et al., 2013). In particular,
lactic acid is a precursor of bio-based materials such as biodegradable
poly-lactic acid (PLA) (Han et al., 2018; Juturu and Wu, 2016). The
global demand for PLA reached 870.8 kilotons in 2016 (Juturu and Wu,
2016), and is predicted to increase by 50 % from 2017 to 2022 (Kong
et al., 2019); thus, resulting in market expansion for lactic acid. Cur-
rently, industrial lactic acid production mainly depends on bio-con-
version of glucose derived from starchy materials to lactic acid via
lactic acid producing strains (Juturu and Wu, 2016), which not only
contributes to most of the total cost of lactic acid fermentation pro-
cesses, but also competes with human food chain. Therefore, utilization
of renewable raw materials for lactic acid production is a highly desired
strategy to meet future market expansion.
At present, sweet sorghum is a preferred feedstock for lactic acid
production owing to the presence of abundant fermentable
carbohydrates (such as glucose, fructose, and sucrose) in its juice (Ou
et al., 2016; Wang et al., 2017, 2016a; Wang et al., 2016b). Moreover,
because of its drought tolerance and saline-alkaline resistance, sweet
sorghum can be grown on marginal lands that are widespread in China
(Dar et al., 2018; Fu et al., 2019). It has been estimated that approxi-
mately 8 million ha of marginal land can be used for the cultivation of
bioenergy crops, including sweet sorghum, in Northern China (Fu et al.,
2019), making sweet sorghum juice more attractive as a low-cost al-
ternative substrate for lactic acid production. In addition, the use of
low-cost nitrogen source (such as dry cell yeast, corn steep liquor,
soybean meal, and cottonseed meal) could be another suitable choice to
further decrease the cost of lactic acid production (Bai et al., 2016;
Kwon et al., 2000; Ooi and Wu, 2015; Tian et al., 2019). Although there
are many reports about the potential use of sweet sorghum juice cou-
pled with low-cost nitrogen sources for lactic acid production, no lactic
acid producer could produce high L-lactic acid titer (> 110 g/L) with
high productivity (> 3.5 g/L/h) in single batch fermentation. To date,
the highest L-lactic acid production by Lactobacillus thermophilus A69
was 114.6 g/L with a productivity of 2.61 g/L/h (Tian et al., 2019).

⁎
Corresponding author at: Department of Biophysics, Institute of Modern Physics, Chinese Academy of Sciences, 509 Nanchang Road, Lanzhou 730000, PR China.
E-mail address: hudongli007@impcas.ac.cn (W. Hu).

https://doi.org/10.1016/j.indcrop.2020.112159
Received 24 September 2019; Received in revised form 16 January 2020; Accepted 20 January 2020
0926-6690/ © 2020 Elsevier B.V. All rights reserved.
X. Tian, et al.
However, the productivity was still inadequate for commercialization
and about 17 g/L residual reducing sugar remained in the fermentation
broth, indicating that other strategies might be combined to further
promote L-lactic acid fermentation efficiency of L. thermophilus A69.
In the past decades, considerable approaches have been employed
to enhance lactic acid fermentation efficiency, such as screening of new
strains (Han et al., 2019a,b), strain improvement by mutation or ra-
tional reconstruction (Jiang et al., 2018; Kong et al., 2019; Lv et al.,
2016; Sun et al., 2015; Upadhyaya et al., 2014; Xu et al., 2010), and
medium optimization (Yu et al., 2008). Besides, application of certain
elicitors, such as vitamin B2, vitamin B3, and vitamin B5, has also been
noted to significantly enhance lactic acid biosynthesis (Han et al.,
2019a,b). Addition of antioxidants, such as vitamin C and sesamol, has
been reported to have the potential to enhance cell growth and fer-
mentation properties of microbial strains by attenuating intracellular
reactive oxygen species (ROS) levels and increasing the antioxidant
capacity (Liu et al., 2015; Ren et al., 2017; Sun et al., 2018). Liu et al.
(2015) demonstrated that addition of sesamol can significantly increase
cell growth and docosahexaenoic acid accumulation in Crypthecodinium
cohnii ATCC 30556 Liu et al., 2015). Ren et al. (2017) found that ad-
dition of 9 g/L vitamin C can promote docosahexaenoic acid accumu-
lation via enhancement of antioxidant capacity and prevention of oxi-
dative damage in Schizochytrium sp. HX-308 (Ren et al., 2017), resulting
in a 30.44 % improvement in docosahexaenoic acid yield. Vitamin C is
an important growth factor and antioxidant, which is known to enhance
total antioxidant capacity (T-AOC) and reduce ROS production in mi-
crobial cells (Ren et al., 2017). In addition to its antioxidation ability,
vitamin C can also improve the activity of enzymes involving in gly-
colytic pathway, such as hexokinase (HK), phosphofructokinase (PFK),
pyruvate kinase (PK), and lactate dehydrogenase (LDH) (Yao et al.,
2018). However, it is still unclear whether external addition of vitamin
C could be useful for improving lactic acid production in L. thermophilus
A69.
In this study, the effects of external vitamin C addition on L-lactic
acid fermentation by L. thermophilus A69 were evaluated, and the
concentration and addition time of vitamin C were optimized. Then, the
impact of vitamin C on key enzyme activities involving in antioxidant
capacity and glycolytic pathway were investigated. Finally, with the
addition of optimal concentration of vitamin C, lab-scale L-lactic acid
fermentation by L. thermophilus A69 was performed using sweet sor-
ghum juice coupled with soybean hydrolysate as feedstocks, and the
biomass, lactic acid productivity, and sugar utilization data were de-
termined.
2. Materials and methods
2.1. Microorganisms, media, and culture conditions
L. thermophilus A69 used in this study was derived from heavy ion
mutagenesis (Jiang et al., 2018), and was cultivated on MRS agar plates
at 50 °C for 24 h, as described in a previous report (Tian et al., 2019).
The seed medium included 20 g/L glucose, 10 g/L peptone, 10 g/L beef
extract, 5 g/L yeast extract, 2 g/L ammonium citrate tribasic, 1 mL/L
Tween 80, 5 g/L sodium acetate, 0.58 g/L MgSO4•7H2O, 2 g/L K2HPO4,
and 0.25 g/L MnSO4•4H2O, with the pH of the medium adjusted to
6.2–6.4. The fermentation medium (shake flask) comprised 100 g/L
glucose, 10 g/L peptone, 10 g/L beef extract, 5 g/L yeast extract, 2 g/L
ammonium citrate tribasic, 1 mL/L Tween 80, 5 g/L sodium acetate,
0.58 g/L MgSO4•7H2O, 2 g/L K2HPO4, 0.25 g/L MnSO4•4H2O, and 50 g/
L CaCO3. The fermentation medium (bioreactor) comprised 126 g/L
clarificated sweet sorghum juice (total reducing sugar), 25 g/L soybean
hydrolysate, 2 g/L ammonium citrate tribasic, 1 mL/L Tween 80, 5 g/L
sodium acetate, 0.58 g/L MgSO4⋅7H2O, 2 g/L K2HPO4, 0.25 g/L
MnSO4⋅4H2O, and 77 g/L CaCO3.
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Industrial Crops & Products 146 (2020) 112159
2.2. Investigation of the effects of vitamin C addition on cell growth and L-
lactic acid production by L. thermophilus A69
L. thermophilus A69 was inoculated into 25 mL of seed medium and
cultured at 50 °C on a ZQLY-180 N rotary incubator (Zhichu Co., Ltd,
Shanghai, China) at an agitation speed of 100 rpm for 8 h.
Subsequently, the cells were inoculated at a 10 % (v/v) inoculum size
into 25 mL of fermentation medium at 50 °C and 100 rpm for 4, 8, 12,
16, 24, 36, and 48 h, respectively. Before addition to the fermentation
medium, vitamin C was configured as 2 M mother liquor and sterilized
by 0.22-μm sterile filtration, and supplemented to fermentation
medium at a final concentration of 50 mM at 0, 4, and 8 h, respectively,
to study the effect of vitamin C addition time on cell growth and L-lactic
acid production. Then, different final concentrations of vitamin C (0,
25, 50, 75, and 100 mM, respectively) were supplemented to fermen-
tation medium at the beginning of fermentation to investigate the effect
of vitamin C concentration on cell growth and L-lactic acid production.
2.3. Determination of key enzymes activities involving in antioxidant
capacity and glycolytic pathway of L. thermophilus A69
L. thermophilus A69 was inoculated into 25 mL of seed medium and
cultured at 50 °C on a ZQLY-180 N rotary incubator (Zhichu Co., Ltd,
Shanghai, China) at an agitation speed of 100 rpm for 8 h.
Subsequently, the cells were inoculated at 10 % (v/v) inoculum size
into 25 mL of fermentation medium (including 0 or 75 mM vitamin C)
at 50 °C and 100 rpm for 4, 8, 12, 24, 36, and 48 h, respectively. Then,
the fermentation broth was centrifuged at 10,000 rpm for 10 min, and
the obtained sediment was washed three times with PBS. After washing,
the cells were disrupted using JY92-IIN ultrasonic disrupter (Xinzhi
Biotechnology Co., Ltd, Ningbo, China), and the intracellular T-AOC,
catalase (CAT), superoxide dismutase (SOD), and total soluble protein
of the cells were determined by using the corresponding assay kits
(Suzhou Keming Bio-Technology Co. ltd., Suzhou, China), according to
the manufacturer’s instructions. The activities of HK, PFK, PK, and LDH
were determined by using the related assay kits (Solarbio
Biotechnology Co., Ltd., Beijing, China).
2.4. Pretreatment of sweet sorghum juice and fermentation with L.
thermophilus A69
Sweet sorghum was harvested in October 2018 in Wuwei, Gansu
Province, China. The pretreatment of sweet sorghum juice was per-
formed as described in a previous report (Liu et al., 2019; Tian et al.,
2019). The fermentation was conducted in a 1-L bioreactor (BLBIO-1GJ,
Lianyungang Bailun Bio-Technology Co. ltd., Jiangsu, China) con-
taining 0.6 L of the fermentation medium (Tian et al., 2019). The seed
culture of L. thermophilus A69 was prepared in 100-mL shake flasks
containing 60 mL of the seed medium at 50 °C and 100 rpm for 8 h.
Then, the seed culture was inoculated into the 1-L bioreactor at
50 °C ± 1 °C and 500 rpm with an aeration rate of 0.2 L/min and 0.08
Mpa of ventilation pressure. The initial pH was automatically con-
trolled at 6.2 ± 0.1 by adding 13.5 M NaOH.
2.5. Analytical methods
The concentration of vitamin C was measured using high perfor-
mance liquid chromatography (HPLC; Ultimate 3000, VWD-3400RS
detector, Thermo Fisher, USA) with a C18 reverse column
(4.6 × 150 mm, 5 μm; Lanzhou Zhongke Kaidi Chemical New-tech Co.,
Ltd., China) containing the mixture of 2.5 g/L metaphosphoric acid and
methyl alcohol (98:2) as a mobile phase at a flow rate of 1 mL/min and
a temperature at 30 °C. Optical purity was measured by HPLC (Ultimate
3000) with a UV detector (254 nm) and ligand exchange column
(Sumichiral CA5000, Sumika Chemical Analysis Service, Osaka, Japan)
containing 1 mM CuSO4 as a mobile phase at a flow rate of 1 mL/min
X. Tian, et al.
and a temperature at 30 °C (Sun et al., 2015). Both glucose and reducing
sugar contents were measured as described by Miller (1959). The fer-
mentation broth was centrifuged and the supernatant was employed to
determine the L-lactic acid concentration using SBA-40D biosensor
analyzer (Tian et al., 2019). The fermentation broth was first treated
with HCl (0.1 mL of broth: 0.7 mL of 0.2 M HCl), and the cell growth
was measured as previously described (Tian et al., 2016). Differences
were evaluated using one-way ANOVA, followed by p value test. Data
are presented as means ± SD (n = 3). Differences were considered
significant at a p value of < 0.05 or 0.01.
3. Results and discussion
3.1. Effects of vitamin C concentration and addition time on cell growth and
L-lactic
acid production by L. Thermophilus A69
As shown in Fig. 1B, L-lactic acid production was enhanced when
vitamin C was added at three different phases after 72 h fermentation
(P < 0.01). In particular, when the vitamin C addition time was 0 h,
the highest L-lactic acid accumulation reached 82.5 g/L at 72 h
(P < 0.01), presenting 16 % enhancement in L-lactic acid production,
when compared with that noted in the control group without vitamin C
supplementation. Similarly, the cell growth and glucose consumption of
L. thermophilus A69 were significantly enhanced when the vitamin C
addition time was 0 h (Fig. 1A and C) (P < 0.01).This indicated that
the optimal addition time of vitamin C was at the beginning of fer-
mentation (0 h).
Previous studies have proved that microbial cells might experience
oxidative stress when transferred from seed culture with nitrogen de-
ficiency to a nutrient-rich fermentation medium (Liu et al., 2012; Ren
et al., 2017). In response to oxidative stress, two distinct types of an-
tioxidative mechanism have been observed in microorganisms: one is
the direct antioxidant activity, which scavenges ROS directly and in-
stantaneously, and the other is mediated indirectly by the synthesis of
3
Industrial Crops & Products 146 (2020) 112159
Fig. 1. The effects of vitamin C addition time
on cell growth and L-lactic acid production. (A)
Cell growth; (B) L-lactic acid concentration; (C)
glucose concentration; Fermentation was car-
ried out in 50 mL flasks containing 25 mL of
fermentation medium at 50 ℃ and 100 rpm.
The error bars in the figure indicate the stan-
dard deviations of three parallel replicates. The
error bars in the figure indicate the standard
deviations of three parallel replicates, and
*p < 0.05 indicated a significant difference
and **p < 0.01 indicated a highly significant
difference.
antioxidant enzymes (Ren et al., 2017). Vitamin C can directly scavenge
ROS by donating its electron and by prevention of oxidative damage in
cells (Molina et al., 2014). In this study, when vitamin C was added at
the beginning of fermentation (0 h), owing to its direct scavenging ac-
tion, the ROS levels remained lower at this stage, when compared with
those noted in the control group without vitamin C supplementation, or
other groups with different vitamin C addition time. Such low ROS level
was beneficial to L. thermophilus A69 metabolism, ultimately leading to
highest cell growth and L-lactic acid accumulation. However, L. ther-
mophilus A69 reached the exponential phase at 4 or 8 h, and although
nitrogen starvation could also induce oxidative stress, the effect was far
less during this phase (Liu et al., 2012; Ren et al., 2017). Microbial cells
in exponential phase can also synthesize numerous stress factors against
free radicals damage induced by nitrogen starvation, which can de-
crease ROS level (Ren et al., 2017). At this stage, addition of vitamin C
into the fermentation broth further reduced ROS level owing to the
scavenging effect of vitamin C. However, excessive decrease in ROS
level was not beneficial to cell growth and L-lactic acid accumulation by
L. thermophilus A69. Similar findings have also been reported by Ren
et al. (2017), who demonstrated that higher ROS level could positively
influence cell growth and lipid accumulation by Schizochytrium sp. This
can explain why the results with addition of vitamin C at 0 h were
better than other addition time groups.
Subsequently, the effect of different concentrations of vitamin C (0,
25, 50, 75, and 100 mM, respectively) on cell growth and L-lactic acid
production by L. thermophilus A69 was examined (Fig. 2). The results
showed that different concentrations of vitamin C addition to fermen-
tation medium promoted L-lactic acid accumulation, glucose con-
sumption, and cell growth profiles of L. thermophilus A69. In particular,
addition of 75 mM vitamin C yielded the highest L-lactic acid accumu-
lation, reaching 81.93 g/L at 48 h, which was 31 % higher than that
noted in the control group without vitamin C supplementation
(P < 0.01) (Fig. 2C). The highest L-lactic acid productivity was also
observed with the addition of 75 mM vitamin C at 48 h (Fig. 2D).
X. Tian, et al.
However, addition of 100 mM vitamin C decreased L-lactic acid accu-
mulation rate, glucose consumption rate, and biomass accumulation
rate of L. thermophilus A69, indicating that high vitamin C concentration
inhibited cell growth and L-lactic acid accumulation by L. thermophilus
A69. Similar results have also been reported in previous studies in
which addition of antioxidants at appropriate concentrations was de-
monstrated to enhance cell growth and fermentation properties of mi-
crobial strains, whereas higher antioxidants concentrations was found
to cause an inhibitory effect (Liu et al., 2015; Ren et al., 2017). Thus,
the optimal concentration of vitamin C was noted to be 75 mM.
3.2. Effects of vitamin C on key enzymes activities involving in antioxidant
capacity and glycolytic pathway of L. Thermophilus A69
To further explore mechanism of the observed effects of vitamin C
on cell growth and L-lactic acid accumulation of L. thermophilus A69, the
intracellular key enzyme activities involving in antioxidant capacity
(such as T-AOC, CAT activity, and SOD activity) of L. thermophilus A69
were determined. T-AOC shows the cell's global resistance to environ-
mental stress. Previous study found that a strong T-AOC played an
important role in essential physiological metabolic process, such as
resisting against membrane-lipid peroxidation and against DNA damage
(Ren et al., 2017). CAT and SOD are known to scavenge ROS such as
O2%– and H2O2 (Sun et al., 2018). As shown in Fig. 3A, the T-AOC va-
lues of the culture with 75 mM vitamin C added at different stages were
significantly higher (P < 0.01) when compared with that noted in the
control group without vitamin C supplementation, indicating that vi-
tamin C considerably improved antioxidant activity as expected. Si-
milar findings have also been proved by Ren et al. (2017). When vi-
tamin C was added into fermentation broth, it can scavenge ROS
directly by donating its electron and by preventing of oxidative damage
in cells (Molina et al., 2014). Vitamin C can also regenerate other an-
tioxidants (such as glutathione, α- tocopherol, and β-carotene) against
ROS damage (Halliwell, 1996; Molina et al., 2014; Palkowitsch et al.,
2011). Thus, ROS level in the group grown with 75 mM vitamin C was
4
Industrial Crops & Products 146 (2020) 112159
Fig. 2. The effects of vitamin C concentration
on cell growth and L-lactic acid production. (A)
Cell growth; (B) Glucose concentration; (C) L-
lactic acid concentration; (D) Productivity.
Fermentation was carried out in 50 mL flasks
containing 25 mL of fermentation medium at
50 ℃ and 100 rpm. The error bars in the figure
indicate the standard deviations of three par-
allel replicates, and *p < 0.05 indicated a
significant difference and **p < 0.01 in-
dicated a highly significant difference.
reduced, and the cells only synthesized low abundance gene expression
of antioxidant enzymes against ROS damage, and lower CAT and SOD
activities were observed (Fig. 3B and C). Based on these results, it can
be concluded that addition of vitamin C could improve intracellular
antioxidant activity, which might be a key reason for the enhancement
of L-lactic acid accumulation in L. thermophilus A69.
It is known that HK, PFK, PK, and LDH are the key enzymes in
glycolytic pathway, which are closely related to lactic acid biosynthesis
in lactic acid bacteria (Juturu and Wu, 2016). HK, PFK, and PK are key
enzymes that convert glucose to pyruvate, and LDH directly metabolize
pyruvate to L-lactic acid. In the present study, the activities of these four
enzymes (HK, PFK, PK, and LDH) at the log phase (4 and 8 h) of L.
thermophilus A69 were examined. When compared with the control
group without vitamin C supplementation, HK, PFK, PK, and LDH ac-
tivities were significantly enhanced with the addition of vitamin C
(Fig. 4), confirming that addition of vitamin C might accelerate meta-
bolic flow from glucose to lactic acid in L. thermophilus A69. In a pre-
vious study, Yao et al. (2018) reported that vitamin C deficient medium
can cause significant reduction in HK, PFK, PK, and LDH activities in
Lactobacillus helveticus CICC 22171. Besides, vitamin C can also con-
siderably influence the expression of genes involved in fatty acid
synthesis, which contributes to high lactic acid accumulation (Yao
et al., 2018).
Collectively, these findings demonstrated that addition of vitamin C
can improve T-AOC and activities of key enzymes involving L-lactic acid
synthesis, which boosted L-lactic acid production in L. thermophilus A69.
3.3. Promotion of L-lactic acid production from sweet sorghum juice and
soybean hydrolysate by vitamin C supplementation
In a previous study, hot lime clarification treatment can boost L-
lactic acid accumulation effectively (Tian et al., 2019). However, hot
lime clarification treatment can affect the composition of sweet sor-
ghum juice including protein, pigment, trace metals (Andrzejewski
et al., 2013). Considering that sweet sorghum juice contained a certain
X. Tian, et al. Industrial Crops & Products 146 (2020) 112159

Fig. 3. Effects of vitamin C on total antioxidant

capacity and key antioxidant enzyme activities.
(A) T-AOC; (B) CAT; (C) SOD. Fermentation
was carried out in 50 mL flasks containing
25 mL of fermentation medium at 50 ℃ and
100 rpm. The error bars in the figure indicate
the standard deviations of three parallel re-
plicates, and *p < 0.05 indicated a significant
difference and **p < 0.01 indicated a highly
significant difference.

amount of vitamin C, we tested the concentration of vitamin C in both vitamin C, indicating that hot lime clarification treatment process al-
crude sweet sorghum juice and clarificated sweet sorghum juice. The lowed the preservation of vitamin C in clarificated sweet sorghum juice.
results showed that crude sweet sorghum juice contained 15.33 mg/L However, such small amount of vitamin C in clarificated sweet sorghum
vitamin C, and clarificated sweet sorghum juice contained 14.83 mg/L juice was not enough for lactic acid fermentation. Thus, we

Fig. 4. Effects of vitamin C on key enzyme ac-

tivities involving glycolytic pathway. (A) hex-
okinase (HK); (B) phosphofructokinase (PFK);
(C) pyruvate kinase (PK); (D) lactate dehy-
drogenase (LDH). Fermentation was carried out
in 50 mL flasks containing 25 mL of fermenta-
tion medium at 50 ℃ and 100 rpm. The error
bars in the figure indicate the standard devia-
tions of three parallel replicates, and
*p < 0.05 indicated a significant difference
and **p < 0.01 indicated a highly significant
difference.

5
X. Tian, et al. Industrial Crops & Products 146 (2020) 112159

Fig. 5. Promoting L-lactic acid production based on sweet sorghum juice and soybean hydrolysate by supplementation of vitamin C. Fermentation was carried out in a
1 L bioreactor containing 0.6 L of sweet sorghum juice.

supplemented 75 mM vitamin C to clarified sweet sorghum juice along
with soybean hydrolysate as a nitrogen source and conducted L-lactic
acid fermentation by L. thermophilus A69 in a 1-L bioreactor. As shown
in Fig. 5, the fermentation terminated at 32 h when 75 mM vitamin C
was added, and a final L-lactic acid concentration of 118.8 g/L with a
productivity of 3.71 g/L/h was achieved by L. thermophilus A69. The
conversion rate of L-lactic acid was 0.97 g/g total reducing sugar, and
only 4.59 g/L of residual reducing sugars remained in the fermentation
broth. When vitamin C was not added into the fermentation medium,
118.3 g/L L-lactic acid was accumulated; however, the fermentation
period extended to 38 h and the productivity was only 3.11 g/L/h.
These results suggested that supplementation of vitamin C further
promoted L-lactic acid accumulation and sugar consumption from sweet
sorghum by L. thermophilus A69.
Table 1 shows a comparison among studies on L-lactic acid accu-
mulation from sweet sorghum juice. In most cases, low L-lactic acid
productivities were achieved by different lactic acid producers when
sweet sorghum juice and low-cost nitrogen sources were used. For ex-
ample, Ou et al. (2016) reported that B. coagulans 36D1 produced L-
lactic acid titer of 126 g L−1 with a low productivity of 0.875 g L−1 h−1
using sweet sorghum juice coupled with corn steep liquor as nitrogen
source. An L-lactic acid titer of 106 g/L with a productivity of 3.31 g/L/
h was obtained by Lactobacillus paracasei No. 8 from sweet sorghum
juice, but expensive yeast extract and peptone as nitrogen sources were
used (Richter and Trager, 1994). From the comparison of the reported
lactic acid production using sweet sorghum juice coupled with low-cost
nitrogen sources, it can be noted that no lactic acid producer could
achieve high L-lactic acid titer (> 118.8 g/L) with high productivity
(> 3.71 g/L/h) in single batch fermentation. In this study, with the
supplement of vitamin C, A69 strain can produce 118.8 g L−1 L-lactic
acid with a productivity of 3.71 g L−1 h−1 using purified sweet sor-
ghum juice coupled with soybean hydrolysate. Hence, L. thermophilus
A69 is commercially more attractive for producing L-lactic acid from
sweet sorghum juice coupled with low-cost nitrogen source owing to its
higher lactic acid producing efficiency. This study also provided a new
solution for enhancement of L-lactic acid fermentation efficiency using
sweet sorghum juice as a feedstock.
Currently, short-term storage of fresh sweet sorghum juice usually
hinders its industrial applications in China. Nevertheless, a combination
of evaporation and chemical preservation has been reported to sig-
nificantly prolong the storage period of sweet sorghum juice with only 5
% fermentable sugar loss (Zhang et al., 2018), making sweet sorghum
juice more attractive for biochemicals production via microbial con-
version. In addition, approximately 8 million ha of marginal land can be
used for sweet sorghum cultivation without competition for land for
food crops (Fu et al., 2019), which can produce about 40.8–84 million
tons of sweet sorghum sugar (Zhao et al., 2009). As a result, in terms of
marginal land utilization for sweet sorghum cultivation, the industrial
application of sweet sorghum juice for the production of biochemicals,
including L-lactic acid, has become highly competitive in China.
4. Conclusion
This work indicated that external vitamin C addition was an effi-
cient and economical approach for enhancement of L-lactic acid fer-
mentation efficiency. The obtained results on key enzyme activities

Table 1
L-Lactic acid production from sweet sorghum juice by lactic acid producers.

Strain Nitrogen source L-Lactic acid Productivity (g/L/h) Lactic acid yield Fermentation process References
(g/L) (g/g)

Lactobacillus paracasei No. 8 Yeast extract and 106 3.31 0.95 Batch Richter and Trager
peptone (1994)
Lactobacillus rhamnosus LA-04-1 Yeast extract 60.25 17.55 0.954 Repeated batch Wang et al. (2016b)
Bacillus coagulans LA1507 & L. rhamnosus Corn steep powder 118 1.84 0.921 Batch Wang et al. (2016a)
LA-04-1
B. coagulans 36D1 Corn steep liquor 126 0.875 0.86 Batch Ou et al. (2016)
Lactobacillus thermophiles A69 Soybean hydrolysate 114.6 2.61 1.02 Batch Tian et al. (2019)
L. thermophilus A69 Soybean hydrolysate 118.8 3.71 0.97 Batch This study

6
X. Tian, et al.
involving antioxidant capacity and glycolytic pathway may help to
further understand the mechanism of vitamin C effect on regulation of
L-lactic acid biosynthesis by L. thermophilus A69. With the specific
supplementation of vitamin C into sweet sorghum juice coupled with
cheaper soybean hydrolysate as the feedstocks, a final L-lactic acid
concentration of 118.8 g/L with the productivity of 3.71 g/L/h was
achieved by A69 strain, which was the highest research report.
CRediT authorship contribution statement
Xuejiao Tian: Methodology, Investigation, Writing - original draft.
Wei Hu: Resources, Methodology, Writing - review & editing,
Supervision, Data curation. Jihong Chen: Resources, Data curation.
Wei Zhang: Validation, Formal analysis, Visualization. Wenjian Li:
Resources, Data curation.
Declaration of Competing Interest
The authors report no ‘conflict of interest’ in connection with this
manuscript.
Acknowledgements
The study was supported financially by the National Natural Science
Foundation of China (No. U1932136), the Western Talents Program of
the Chinese Academy of Sciences (Y706030XB0). Partially supported by
Open Funds of Key Laboratory of Extreme Environmental Microbial
Resources and Engineering, Gansu Province (EEMRE201801).
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