Process Biochemistry xxx (xxxx) xxx–xxx

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Process Biochemistry
journal homepage: www.elsevier.com/locate/procbio

Efficient production of lactic acid from sugarcane molasses by a newly

microbial consortium CEE-DL15
Yaqin Sun, Zhenzhen Xu, Yafeng Zheng, Jinjie Zhou, Zhilong Xiu
⁎

School of Life Science and Biotechnology, Dalian University of Technology, PR China

ARTICLE INFO ABSTRACT

Keywords: Sugarcane molasses, a waste from sugar manufacturing processes, has promising future to be utilized as a cheap
Microbial carbon source for lactic acid production. In this study, a newly microbial consortium, CEE-DL15, for the con-
Consortium version of sugarcane molasses to lactic acid was selected and evaluated. The consortium was screened from cattle
Lactic acid stomach content and mainly consisted of Clostridium sensustricto (57.29%), Escherichia (34.22%), and
Sugarcane molasses
Enterococcus (5.32%). Lactic acid production was explored under the deficiency of sterilization and molasses
Corn steep liquor powder
acidification, with corn steep liquor powder used as organic nitrogen source. In batch fermentations using su-
garcane molasses of 350 g/L and corn steep liquor powder of 18.5 g/L without additional nutrients, CEE-DL15
produced 112.34 g/L lactic acid (107.40 g/L L-lactic acid and 4.94 g/L D-lactic acid), with a yield of 0.81 g/g and
a maximum productivity of 4.49 g/(L.h), which is the best lactic acid productivity from molasses published so
far. Economic analysis indicated that lactic acid fermentation cost was only 448 USD/ton using molasses and
corn steep liquor powder, which was only 37.7% of the total cost when compared with glucose as carbon source
and MRS medium as nitrogen source. This work demonstrated that the high adaptation to molasses of microbial
consortium CEE-DL15 might be a promising alternative for the economical production of lactic acid.

1. Introduction
Lactic acid is an important platform chemical that has been applied
in foods, cosmetics, textiles, pharmaceuticals and many other industrial
fields. Biotechnological lactic acid production has several advantages
over chemical approaches regarding environmental issues, good utili-
zation of diverse substrates, and the feasibility to produce optically pure
lactic acid forms [1]. However, higher production costs have hindered
the large-scale application of lactic acid. Therefore, reduction of lactic
acid production cost through utilization of inexpensive substrate and
improvement of lactic acid production and productivity has become an
important goal [2].
The carbon source in lactic acid fermentation is responsible for the
major factors in the economic production of lactic acid. Studies on
lactic acid fermentation from raw materials as fermentative substrates
have suggested numerous candidates, such as sugarcane molasses [3,4],
paper sludge [5], cellulose [6], apple pomace [7], kitchen wastes [8],
Jerusalem artichoke powder [9], etc. As a relatively cheap and abun-
dant raw material, sugarcane molasses is a by-product prepared from
the liquid waste of sugar production and the output of sugarcane mo-
lasses in China is about 3 million tons annually, which makes it an
available raw material for microbial production [10]. Sugarcane mo-
lasses contains about 50% (w/v) total sugar (sucrose, glucose, and
fructose), 0.5−0.9% (w/v) nitrogen, 10% (w/v) inorganic salts, etc
[11,12]. However, some hazardous substances such as 5-hydro-
xymethylfurfural and excessive metallic ions are generated during
sugar manufacturing process, which are toxic to cell growth, causing
low conversion yield and productivity [13]. To eliminate the inhibition
of hazardous substances and improve sugarcane molasses utilization,
genetic engineering strains or mutagenic strains were applied to lactic
acid production [14,15]. Escherichia coli WYZ-L, enhancing expression
of the sucrose operon (cscA and cscKB), produced 75 g/L of lactic acid,
with a yield of 0.85%, and a maximum productivity of 1.18 g/(L.h)
using sugarcane molasses and corn steep liquor without additional
nutrients [15]. High lactic acid concentration of 166 g/L was obtained
at a molasses sugar concentration of 190 g/L with a productivity of
4.15 g/(L.h) by Lactobacillus delbrueckii mutant Uc-3, isolating by UV
mutagenesis [14]. Moreover, co-feeding strategy based on the utiliza-
tion of cane molasses/glucose carbon sources was considered to im-
prove the utilization of cane molasses. A titer of 168.3 g/L L-lactic acid
was obtained by a Bacillus coagulans strain H-1 after 78 h fed-batch
fermentation, with a productivity of 2.1 g/(L.h) and a yield of 0.88 g/g

⁎
Corresponding author at: School of Life Science and Biotechnology, Dalian University of Technology, No. 2 Linggong Road, Ganjingzi District, Dalian City,
Liaoning Province, 116024, PR China.
E-mail address: zhlxiu@dlut.edu.cn (Z. Xiu).

https://doi.org/10.1016/j.procbio.2019.03.022
Received 31 October 2018; Received in revised form 7 February 2019; Accepted 24 March 2019
1359-5113/ © 2019 Elsevier Ltd. All rights reserved.

Please cite this article as: Yaqin Sun, et al., Process Biochemistry, https://doi.org/10.1016/j.procbio.2019.03.022
Y. Sun, et al. Process Biochemistry xxx (xxxx) xxx–xxx

[4]. Although there were many efforts to improve utilization of su-

garcane molasses, the productivity was still not too high due to the
inhibition of the impurities in cane molasses. Cane molasses contains a
number of inorganic and organic inhibitors, which may impede the
metabolism of cells. Therefore, cane molasses was usually used in fer-
mentation after pretreatment to reduce the level of various inhibitors
before application in fermentation. However, pretreatment will raise
the fermentation cost of cane molasses and increase workload. If no
pretreatment of cane molasses is allowed, it would be undoubtedly
economical for fermentation. Moreover, open fermentation is a great
advantage for bio-chemicals production while autoclaving would col-
orize the broth, lead to sugar losses and increase production cost. If
these operation steps are reduced, a large amount of energy could be
saved for economical lactic acid production using raw materials.
Currently, almost all commercial lactic acid production is based on
carbohydrate fermentations using pure cultures of lactic acid bacteria
Fig. 1. Lactic acid production under different operation conditions at initial
[16]. In contrast to pure culture fermentations, microbial consortium
sugarcane molasses concentration of 250 g/L. Fermentation was carried out at
offers the advantages of no requirement of sterilization, versatile fer- 37 °C in 250 mL serum bottle containing 100 mL medium under anaerobic
mentation conditions and utilization of complex feedstock. Liang et al conditions. MRS medium was selected and fermentation was complete at 48 h.
demonstrated the feasibility of lactic acid production from commercial Values are means of three independent fermentations.
potato peel waste by mixed culture fermentations operated in batch and
sequencing batch modes [17]. The highest volumetric productivity of
components [26]: 30 g/L glucose, 2.5 g/L yeast extract, 5 g/L peptone,
lactic acid was achieved in a continuous stirred-tank reactor (CSTR) by
5 g/L beef extract, 2 g/L sodium acetate, 2 g/L ammonium citrate,
microbial community [16]. Although the final lactic acid concentration
0.2 g/L MgSO4, 0.05 g/L MnSO4. The medium was sterilized before
was not high, these researches implied that microbial consortium might
incubation.
be an alternative to pure strains for efficient degradation of complex
Fermentation medium (MRS medium) used for lactic acid produc-
substrates with multiple impurities, which has characteristics of flexible
tion contained the following components [26]: 5 g/L yeast extract,
metabolism and low requirement for operation. Up to now, microbial
10 g/L peptone, 10 g/L beef extract, 5 g/L sodium acetate, 2 g/L
consortium is applied to bio-chemicals production, e.g. 1,3-propanediol
K2HPO4, 2 g/L ammonium citrate, 0.58 g/L MgSO4, 0.25 g/L MnSO4,
[18–21] and bioenergy, e.g. ethanol [22], butanol [23] and hydrogen
1 mL tween 80. Sugarcane molasses was used as sole carbon source.
[24]. In these studies, microbial consortium exhibits advantages such as
MRS medium was sterilized or not according to experiment design as
high adaptation to raw materials, simple requirement for nutrients, and
shown in Fig. 1.
unaffected performance under non-sterile conditions.
Simplified MRS (S-MRS) medium contained the following compo-
The aim of this study was to evaluate a microbial consortium for
nents: 2.5 g/L yeast extract, 5 g/L peptone, 5 g/L beef extract, 5 g/L
efficient conversion of sugarcane molasses to lactic acid under open
sodium acetate, 2 g/L K2HPO4, 2 g/L ammonium citrate, 0.58 g/L
operation conditions. Microbial consortium CEE-DL15 was isolated,
MgSO4, 0.25 g/L MnSO4. Sugarcane molasses was used as sole carbon
which could convert sugarcane molasses to lactic acid efficiently. The
source. The medium was not sterilized before incubation.
physiological and biochemical characteristics of CEE-DL15 were de-
CSLP medium contains 18.5 g/L of CSLP. It was not sterilized before
termined through 16S rRNA gene analysis. Then batch fermentation
incubation.
was carried out to evaluate the consortium adaptation to cane molasses
without sterilization and acidification. Meanwhile, the microbial com-
munity structures of CEE-DL15 during the fermentation process were 2.3. Enrichment of microorganisms
also analyzed and illustrated. Finally, economic evaluation was devel-
oped and compared among different research routes to give insight of Cattle stomach was obtained from Dalian slaughter house and all
lactic acid production. the samples were stored at −20 °C for further studies. Stomach content
was prepared by adding 100 g fresh content of cattle stomach into
2. Materials and methods 100 mL saline solution, mixing for 2 min with a vortex, and then fil-
tering by sterile gauze. The filtrate medium was stomach content sus-
2.1. Chemicals pended matter. About 2 g of stomach content or 2 mL stomach content
suspended matter was inoculated to a 250 mL flask sealed with cotton
Sugarcane molasses containing a sucrose content of 28.71% (w/v), cap or a 250 mL serum bottle containing 100 mL enrichment medium at
fructose content of 10.35% (w/v) and glucose content of 3.77% (w/v), 37 °C and 200 rpm. The enrichment medium was sterilized before in-
was purchase from Liuzhou, China. Corn steep liquor powder (CSLP) cubation. After 12 h incubation, 2 mL of the culture broth was trans-
with a nitrogen content of 9% (w/v) was afforded by Yuancheng bio- ferred to another flask or serum bottle with the same enrichment
technology company, Liaoning, China. All the medium containing su- medium and incubated for 12 h. This process was repeated ten times.
garcane molasses were freshly prepared and used for fermentation. All
other chemicals were of analytical grade and commercially available. 2.4. Batch fermentation in 5 L bioreactor

2.2. Medium In the experiments of operation conditions comparison, the su-

Enrichment medium used for bacterial enrichment contains the
following components [25]: 40 g/L glucose, 5 g/L yeast extract, 5 g/L
sodium fumarate, 0.1 g/L monensin, 3 g/L K2HPO4, 1 g/L NaCl, 1 g/L
(NH4)2SO4, 0.5 g/L CaCl2, 0.5 g/L MgCO3, 0.5 g/L MgCl2. The enrich-
ment medium was sterilized before incubation.
Seed medium used for seed culture contains the following
garcane molasses was pretreated by acid according to the published
method [14], while it was used directly without pretreatment in the
batch and fed-batch fermentation experiments.
The batch fermentation was carried out in a 5 L bioreactor con-
taining 2 L fermentation medium at 37 °C and 200 rpm. The inoculum
volume was 5% (v/v).
The pH was controlled at 6.3 by automatic addition of 5 mol/L

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Y. Sun, et al.
NaOH for the batch fermentation in a 5 L bioreactor. Samples were
taken periodically to determine the concentration of lactic acid, by-
products and reducing sugar. An anaerobic environment in the bior-
eactor was produced by sparging with nitrogen gas at 0.1 vvm for 0.5 h
before inoculation and 3.5 h after inoculation, respectively.
2.5. Analytical methods
The biomass concentration was measured at 660 nm with the fer-
mentation medium as blank. Samples were pretreated by centrifugation
at 9720 × g for 10 min, and the supernatants were diluted with deio-
nized water to the desired extent. Glucose, fructose and sucrose con-
centrations were analyzed by high performance liquid chromatography
(HPLC) equipped with a HypersilNH2-S column with a column tem-
perature of 30 °C. Acetonitrile and water (75:25, v/v) was the mobile
phase with a flow rate of 0.6 mL/min. Lactic acid and ethanol con-
centrations were analyzed by HPLC equipped with an Aminex HPX-
87Hcolumn with a column temperature of 65 °C. The mobile phase was
5 mM H2SO4 with a flow rate of 0.6 mL/min. Other acids concentrations
were analyzed by HPLC using a C18 column at 214 nm with an ultra-
violet detector, 2‰ phosphoric acid and acetonitrile (96.5:3.5, v/v) as
the mobile phase at a flow rate of 1 mL/min. Isomer of lactic acid was
analyzed by HPLC using a chiral column of Chirex 3126 (D)-penicillami
with a PDA detector at 254 nm, 2 mM CuSO4 and isopropyl alcohol-
water (95:5, v/v) as the mobile phase at a flow rate of 0.7 mL/min [27].
2.6. Composition analysis of microbial consortium CEE-DL15
The bacterial community composition of microbial consortium CEE-
DL15 was studied by 16S rRNA gene amplicon high-throughput se-
quencing. Total DNA of selected microbial consortium was extracted by
miniBEST bacterial genomic DNA extraction kit (Takara, Ver 3.0) and
sequenced on IlluminaHiSeq 2000 platform by Sangon Biotech in
Shanghai, China. 16S rRNA gene sequences of the consortia have been
submitted to the NCBI Sequence Read Archive (SRA) under accession
number SRP127781.
3. Results and discussion
3.1. Evaluation of microbial consortium
Different microbial consortiums for conversion of glucose to lactic
acid were screened out from the stomach. Twenty different microbial
consortia from the same cattle’s stomach were chosen to determine
their ability to convert glucose to lactic acid. Four enrichment and
evaluation were investigated and the optimum results are presented in
Table 1. Considering anaerobic characteristic of stomach environment,
only anaerobic fermentation was developed for microbial consortia.
The highest lactic acid concentration (41.80 g/L) and highest yield
(0.90 g/g) were obtained by microbial consortium CEE-DL15 from
stomach content under anaerobic enrichment and anaerobic fermen-
tation, respectively. It was thus chosen as the microbial consortium for
subsequent investigation.
Table 1
Enrichment and evaluation of microbial consortium for the coversion of glucose
to lactic acid.
Origin Enrichment Fermentation Lactic acid
(g/L)
1 Stomach content anaerobic anaerobic 40.95
suspended matter
2 Stomach content aerobic anaerobic 35.67
suspended matter
3 Stomach content anaerobic anaerobic 41.80
4 Stomach content aerobic anaerobic 29.76
Process Biochemistry xxx (xxxx) xxx–xxx
Table 2
Microbial community analysis of the original consortium of CEE-
DL15.
Taxonomy Percentage (%)
Clostridium sensu stricto 57.29
Escherichia 34.22
Enterococcus 5.32
Anaerobacter 1.94
Melissococcus 0.18
Exiguobacterium 0.14
Unclassified 0.91
3.2. Abundance of microbial consortium CEE-DL15
16S rRNA gene amplicon high-throughput sequencing was per-
formed to investigate the bacterial composition of microbial consortium
CEE-DL15 and the result was presented in Table 2. A total of 62577
good quality sequence reads were generated and the read utilization
ratio was 98.64%. Four different bacterial families were identified. As
shown in Table 2, the most abundant family type found in microbial
consortium CEE-DL15 belonged to Clostridium sensu stricto (57.29%)
and Escherichia (34.22%), followed by Enterococcus (5.32%), Anaero-
bacter (1.94%), and others (1.23%).
3.3. Utilization of sugarcane molasses under different operation conditions
by microbial consortium CEE-DL15
Sugarcane molasses contains a number of inorganic and organic
inhibitors, which may impede the metabolism of cells. Therefore, it is
generally used as pretreated to reduce the level of various inhibitors
before application in fermentation. However, pretreatment will raise
the cost of fermentation of molasses and increase workload [4].
Moreover, in a previous study, sugarcane molasses and the fermenta-
tion medium was generally autoclaved which consumed a large amount
of energy, colorized the broth, and lead to sugar losses. The char-
acteristic of open fermentation ability owned by microbial consortium
CEE-DL15 makes the fermentation medium be utilized without auto-
claving.
In this study, sugarcane molasses utilization under sterilization and
acidification conditions compared with no sterilization and no acid-
ification was investigated under different initial sugarcane molasses
concentration of 100 g/L, 200 g/L and 250 g/L, respectively. Lactic acid
concentration, yield and productivity from sugarcane molasses via non-
sterile and non-pretreated process under 250 g/L of sugarcane molasses
was compared as Fig. 1 shown. The results showed that the microbial
consortium can grow well without sterilization and acidification. Lactic
acid concentration, yield and productivity were a bit high in this con-
dition than in sterilization and acidification, implying that the non-
sterile and non-pretreated fermentation was feasible for conversion of
sugarcane molasses to lactic acid. A similar tendency was obtained at
100 g/L and 200 g/L of sugarcane molasses under sterilization and
acidification conditions, respectively, compared with no sterilization
and acidification. For example, a titer of 35.48 g/L lactic acid, a yield of
0.71 g/g, and a productivity of 1.47 g/(L.h) were obtained using 100 g/
L sugarcane molasses under sterilization and acidification. In contrast, a
titer of 36.71 g/L lactic acid, a yield of 0.74 g/g, and a productivity of
1.53 g/(L.h) were obtained under no acidification and sterilization.
Yield Therefore, to reduce the cost of lactic acid production and work-
(g/g) load, sugarcane molasses was directly utilized without any pretreat-
ment and sterilization during fermentation process.
0.88
0.77 3.4. Selection of economical and simplified fermentation medium
0.90
0.65 Organic nitrogen source containing amino acids, nucleotides, and
vitamins are important and essential for cell growth. Though sugarcane
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Y. Sun, et al. Process Biochemistry xxx (xxxx) xxx–xxx

Fig. 2. Effects of medium composition on conversion of sugarcane molasses to

lactic acid at initial sugarcane molasses concentration of 250 g/L. Fermentation
Fig. 3. The tolerance of microbial consortium CEE-DL15 to sugarcane molasses
was carried out at 37 °C and 200 rpm in 250 mL serum bottle containing 100 mL
in batch fermentations in 5 L bioreactor. CSLP medium was selected.
medium under anaerobic conditions. MRS, S-MRS and CSLP medium were se-
Fermentation was carried out at 37 °C, pH 6.3 and 200 rpm. CSLP medium was
lected. The fermentation for MRS, S-MRS and CSLP medium was complete at
selected without sterilization. (A) Lactic acid concentration. (B) Microbial
48 h, 42 h, and 36 h, respectively. Values are means of three independent fer-
growth. (C) Fermented sugar concentration.
mentations.

molasses already contains some nitrogen nutrients, an addition of other

organic nitrogen source may enhance the fermentation courses.
Selecting an economical and simplified fermentation medium was im-
portant for large-scale lactic acid production.
In this study, three different medium were compared and studied for
conversion of sugarcane molasses to lactic acid. MRS medium including
5 g/L of yeast extract powder, 10 g/L of beef extract, 10 g/L of peptone
and other salts listed in section 2.2 was selected according to a previous
report [26]. Simplified MRS medium included 2.5 g/L of yeast extract
powder, 5 g/L of beef extract, 5 g/L of peptone and other salts. CSLP
medium only included 18.5 g/L corn steep liquor powder without ad-
ditional nutrients according to the organic nitrogen contents in MRS
medium. The results of the influence of three medium over the pro-
duction and productivity of lactic acid are displayed in Fig. 2. Among
the three fermentation medium evaluated, CSLP medium achieved the
highest lactic acid productivity (2.31 g/(L.h)) and a little low lactic acid
concentration and yield (83.24 g/Land 72%, respectively). This in-
dicated that CSLP addition enhanced cell growth while sugarcane mo-
lasses consumption and lactic acid formation rate increased. The pri-
mary costs associated with lactic acid production include the
fermentative substrates of nutrients, nitrogen sources and sugars re-
quired for cell growth. Considering the price of yeast extract, MRS
medium would not be economical to for application on a large-scale
lactic acid production. According to our investigated results, corn steep
liquor powder with sugarcane molasses could be used for the produc-
tion of lactic acid without additional nutrients. The unit price of CSLP
was only around 5–10% of the unit total price of yeast extract powder
and peptone. Approximately 90% of nitrogen sources costs can be re-
duced while CSLP medium instead of traditional MRS medium. Fig. 4. Time course of batch fermentation by microbial consortium CEE-DL15
at initial sugarcane molasses concentration of 500 g/L in 5 L bioreactor.
Fermentation was carried out at 37 °C, pH 6.3 and 200 rpm. CSLP medium was
3.5. Sugarcane molasses tolerance of microbial consortium CEE-DL15 in selected without sterilization. (A) Fermented sugar and lactic acid concentra-
batch fermentations tion. (B) Sucrose, fructose and glucose concentration. (C) By-products con-
centration.
The capability of sugarcane molasses utilization by microbial con-
sortium CEE-DL15 was evaluated with different molasses concentra-
14.41 g/L sucrose was remained and 130.89 g/L lactic acid was pro-
tions (250–500 g/L sugarcane molasses). As shown in Fig. 3, microbial
duced with a productivity of 0.90 g/(L.h) and a yield of 73%. Total
consortium CEE-DL15 could tolerate high concentration of molasses up
consumption of glucose and fructose was in 27 h and 105 h, respectively
to 500 g/L. Cell growth was severely inhibited when sugarcane mo-
(Fig.4). The results showed that microbial consortium CEE-DL15 had
lasses concentration was increased to 400 g/L. Sugar consumption and
excellent tolerance to sugarcane molasses. The final byproduct con-
lactic acid formation was reduced.
centrations of succinic acid, acetic acid, formic acid and ethanol were
When 500 g/L sugarcane molasses was utilized, cell growth was
5.38 g/L, 3.75 g/L, 2.15 g/L and 1.75 g/L, respectively.
constant for about 120 h (from 25 h to 145 h). In the end, about

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Y. Sun, et al. Process Biochemistry xxx (xxxx) xxx–xxx

Table 3
The concentration of lactic acid isomers at different sugarcane molasses con-
centrations.
Samples Sugarcane Fermentation Total L-lactic D-lactic L-lactic
molasses(g/L) time (h) lactic acid acid acid
acid (g/L) (g/L) (g/L) (%)

1 250 5 28.66 25.11 3.65 87.61

2 250 19 85.95 78.42 7.53 91.24
3 350 25 112.34 107.4 4.94 95.60
4 400 40 36.52 31.26 5.26 85.60
5 500 40 28.32 23.85 4.47 84.22

Five samples for optical purity of lactic acid at different molasses

concentrations were analyzed by College of Light Industry and Food
Science, South China University of Technology. The concentration of
lactic acid isomers at different sugarcane molasses concentrations was
shown in Table 3.
3.6. Fermentation performance of microbial consortium CEE-DL15 and
economic analysis
Lactic acid production from sugarcane molasses and corn steep li-
quor powder without additional nutrients were evaluated under batch
fermentation in 5-L bioreactor. Fig. 5 illustrated the time courses of
batch fermentation of 350 g/L of sugarcane molasses. In 350 g/L of
sugarcane molasses batch fermentation, 112.34 g/L of lactic acid
(107.40 g/L L-lactic acid and 4.94 g/L D-lactic acid) was obtained after
25 h fermentation with a high productivity of 4.49 g/(L.h) and a yield of
81%. To our knowledge, this is the highest productivity ever reported
Fig. 5. Time course of batch fermentation by microbial consortium CEE-DL15
at initial sugarcane molasses concentration of 350 g/L in 5 L bioreactor.
Fermentation was carried out at 37 °C, pH 6.3 and 200 rpm. CSLP medium was
selected without sterilization. (A) Fermented sugar and lactic acid concentra-
tion. (B) Sucrose, fructose and glucose concentration. (C) By-products con-
centration.
Fig. 6. The bacterial community structure of CEE-DL15 during the fermentation
at initial sugarcane molasses concentration of 350 g/L in 5 L bioreactor.
for lactic acid production for sugarcane molasses utilization.
For 350 g/L of sugarcane molasses batch fermentation, 15.76 g/L of
glucose, 34.13 g/L of fructose and 88.56 g/L of sucrose were consumed
in 7.5 h, 12 h and 25 h, respectively. The consumption rates of glucose,
fructose and sucrose in sugarcane molasses were 2.10 g/(L.h), 2.84 g/
(L.h) and 3.54 g/(L.h), respectively. A high consumption rate of redu-
cing sugar was obtained in sugarcane molasses fermentation by mi-
crobial consortium CEE-DL15. The final concentrations of byproducts of
succinic acid, acetic acid, formic acid and ethanol were 4.23 g/L,
3.44 g/L, 2.15 g/L and 2.42 g/L, respectively.
Fig. 6 showed changes in microbial community structures of CEE-
DL15 during the fermentation process at initial sugarcane molasses
concentration of 350 g/L. the microbial community was not con-
servative at the interspecific level during the total cultivation condi-
tions. For the preliminary stage, the microbial community structures in
sugarcane molasses were similar to its cultivation using glucose as
carbon source shown in Table 2. When fermentation was developed to
log phase (8 h), Escherichia became the dominant bacteria with a pro-
portion of 79.46%. The proportion of Clostridium sensu stricto dropped
to 13.92%, while the abundance of Enterococcus increased to 6.1%. At
the end of the fermentation (24 h), Escherichia and Enterococcus in-
creased continually to 83.3% and 13.32%, respectively. Lactobacillus
which was not detected either in microbial consortium of CEE-DL15
using glucose as carbon source or at the preliminary stage of CEE-DL15
using sugarcane molasses as carbon source, accounted for 2.21%. As
shown in Fig. 5, glucose was exhausted in 7.5 h, while 14.5 g/L of
fructose and 65.78 g/L of sucrose still remained after 8 h. The abun-
dance changes of microbial community structures might imply that
Clostridium sensu stricto was apt to utilize glucose, while Escherichia was
apt to utilize sucrose. The complicated community interactions between
the different microorganisms in co-culture systems need to be further
studied in our subsequent work.
Sugarcane molasses was utilized for lactic acid fermentation in
previous report and some of them presented noticeable results.
According to the study by Calabia and Tokiwa [28], 107 g/L lactic acid
with a productivity of 1.48 g/(L.h) was obtained from sugarcane mo-
lasses under MRS medium by Lactobacillus delbrueckii. To avoid su-
garcane molasses inhibition, co-feeding regulation strategy was applied
for lactic acid production. The co-feeding strategy using cane molasses
and glucose will supply sufficient carbon sources for lactic acid fer-
mentation without inducing too high concentration of hazardous sub-
stances. 168.3 g/L L-lactic acid was obtained by a Bacillus coagulans
strain H-1 with a productivity of 2.1 g/(L.h) and a yield of 88%, using a
co-feeding strategy based on the utilization of the mixture containing
149 g/L cane molasses and 185 g/L glucose [4]. Moreover, mutant and
genetic strains were evolved and screened out to improve molasses
utilization and productivity. 75 g/L lactic acid with a yield of 85%, and
a productivity of 1.18 g/(L.h) was produced by E. coli WYZ-L which was

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Y. Sun, et al. Process Biochemistry xxx (xxxx) xxx–xxx

Table 4
Costs analysis and comparison for carbon source, nitrogen source and sterilization.* *Costs and energy consumption were per ton lactic acid produced.
References Lactic acid Carbon source Nitrogen source Sterilization Material and
(US dollar $) (US dollar $) sterilization
Concentration Yield Productivity Energy Cost (US dollar $)
(g/L) (g/g) (g/(L.h)) consumption (US dollar $)
(106 kJ)

[29] 192.0 0.96 3.99 Glucose 521 yeast extract (5 g/L), peptone 619 1.97 49 1189
(10 g/L),
beef extract (10 g/L)
[14] 166.0 0.95 4.20 Molasses 263 yeast extract (5 g/L) 179 2.28 57 499
[4] 165.7 0.92 1.60 Glucose 546 CSLP (1 g/L) 3 2.28 57 606
168.3 0.88 2.10 Glucose/ 446 CSLP (1 g/L) 3 2.25 56 505
Molasses
[15] 75.0 0.85 1.18 Molasses 294 CSLP -a 5.04 125 419b
[28] 107.0 0.90 1.48 Molasses 278 yeast extract (5 g/L), peptone 619 3.53 88 985
(10 g/L),
beef extract (10 g/L)
This study 112.3 0.81 4.49 Molasses 375 CSLP (18.5 g/L) 73 0 0 448

Costs and energy consumption were per ton lactic acid produced.
a
The mass of CSLP was not given.
b
The cost of nitrogen source was not included.

an enhanced expression of the sucrose operon (cscA and cscKB) [15].
High concentration of lactic acid (166 g/L) was obtained with a pro-
ductivity of 4.2 g/(L.h) by Lactobacillus delbrueckii mutant Uc-3 [14].
The mutant was isolated by UV mutagenesis followed by selection on
the basis of a bigger zone of acid formation on sucrose-based medium.
In their studies, sugarcane molasses was pretreated by hydrolysis and
5 g/L yeast extract was used to supply nitrogen. It is noteworthy that
only CSLP was required to obtain high lactic acid productivity even at a
high molasses concentration according to our work. A maximum pro-
ductivity of 4.49 g/(L.h) was obtained, which is the best lactic acid
productivity from molasses published so far.
According to previous studies, the primary cost of lactic acid pro-
duction includes fermentative substrates of nutrients, nitrogen sources
and carbon sources [2]. In this study, the cost of carbon sources and
nitrogen sources was analyzed to evaluate the batch fermentation for
lactic acid production by microbial consortia CEE-DL15. The best re-
ported results of lactic acid production from glucose and molasses were
selected for comparison with our research [14,29] and the results were
presented in Table 4.
The cost of carbon and nitrogen sources was defined as
Ci Pi
CLA
( i = cs, ns ) , where Ccs , Cns and CLA represent the concentrations of
carbon source, nitrogen sources in medium and lactic acid produced,
respectively. Pcs and Pns represent the prices of carbon source and ni-
trogen source. Costs and energy consumption were per ton lactic acid
produced. The prices of glucose and cane molasses were approximately
500 USD/ton and 120 USD/ton, respectively. The prices of nitrogen
sources in MRS medium consisting of yeast extract, peptone and beef
extract were approximatly 5944 USD/ton, 4458 USD/ton and 4458
USD/ton, respectively. The price of corn steep liquor powder in our
study was approximately 446 USD/ton. Moreover, the cost of ster-
ilization was also analyzed and evaluated. The energy consumption of
sterilization was defined as Q = C*M*Δt, where C, M, andΔt represent
the specific heat of fermentation broth, mass of fermentation broth and
temperature difference, respectively. For simplicity, the specific heat of
fermentation broth was assigned as 4.2 kJ/(kg.oC), the specific heat of
water. Costs and energy consumption in Table 4 were per ton of re-
quired lactic acid produced.
When using cane molasses and glucose as co-feeding carbon sources,
the production cost was 83% compared to using glucose as the solo
carbon source in the study of Xu et al [4]. Although the low pro-
ductivity was obtained in their study, cane molasses used in this study
was not pretreated, and the organic nitrogen in the fermentation
medium was only 1 g/L corn steep powder. The cost analysis and
comparison for carbon source, nitrogen source and sterilization in
Table 4 showed that the cost of cane molasses occupied almost 50–60%
portion of the cost of glucose in different studies [14,28,29]. The cost
for MRS medium including yeast extract, peptone and beef extract oc-
cupied over 50% portion of total cost for materials and sterilization
[28,29], while the cost of CSLP was only 10% compared to using MRS
as nitrogen source. Economic analysis indicated that lactic acid pro-
duction cost was 1189 USD/ton using glucose as carbon source and
MRS medium as nitrogen source. Lactic acid production cost was 499
USD/ton using molasses as carbon source and yeast extract as nitrogen
source by Lactobacillus delbrueckii subsp. delbrueckii mutant Uc-3 in
batch fermentation. In our study, lactic acid production cost was only
448 USD/ton using molasses as carbon source and CSLP as nitrogen
source by microbial consortium CEE-DL15, which is only 37.7% of the
cost when using glucose as the solo carbon source and MRS medium as
nitrogen source. Therefore, lactic acid production strategy from su-
garcane molasses and CSLP by microbial consortium CEE-DL15 is pre-
ferable on an economical viewpoint.
It should be pointed out that sugarcane molasses should be con-
trolled by acidification according to the study of Dumbrepatil A et al
[14]. Cane molasses contains a number of inorganic and organic in-
hibitors, which may impede the metabolism of cells. Therefore, cane
molasses needs to be pretreated to reduce the level of various inhibitors.
However, pretreatment will raise the fermentation cost of cane mo-
lasses and increase workload. In the present study, sugarcane molasses
was used without any pretreatment, which might be due to the strong
tolerance of microbial consortium CEE-DL15. Moreover, the char-
acteristic of open fermentation ability owned by microbial consortium
CEE-DL15 makes the fermentation medium be utilized without auto-
claving.
4. Conclusion
In this study, we successfully achieved high production of lactic acid
and high tolerance of molasses with a selected microbial consortium
CEE-DL15. The evaluation of CEE-DL15 has the potential for cost-ef-
fective production of lactic acid using CSLP as medium without addi-
tional nutrients supplements. 112.34 g/L Lactic acid (107.40 g/L L-
lactic acid and 4.94 g/L D-lactic acid), with a yield of 0.81 g/g, a pro-
ductivity of 4.49 g/(L.h), which is the best lactic acid productivity from
molasses published so far was obtained when 350 g/L sugarcane mo-
lasses were utilized without any pretreatment and sterilization. This
study provides an efficient and economical option for the utilization of
raw materials in microbial production.

6
Y. Sun, et al.
Acknowledgements
This work was supported by the National Natural Science
Foundation of China (Grant Nos. 21476042 and 21306021) and the
Fundamental Research Funds for Central Universities of China
(DUT17ZD209).
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