Accepted Manuscript

Title: Efficient production of lactic acid from sugarcane

molasses by a newly microbial consortium CEE-DL15

Authors: Yaqin Sun, Zhenzhen Xu, Yafeng Zheng, Jinjie

Zhou, Zhilong Xiu

PII: S1359-5113(18)31640-4
DOI: https://doi.org/10.1016/j.procbio.2019.03.022
Reference: PRBI 11611

To appear in: Process Biochemistry

Received date: 31 October 2018

Revised date: 7 February 2019
Accepted date: 24 March 2019

Please cite this article as: Sun Y, Xu Z, Zheng Y, Zhou J, Xiu Z, Efficient production
of lactic acid from sugarcane molasses by a newly microbial consortium CEE-DL15,
Process Biochemistry (2019), https://doi.org/10.1016/j.procbio.2019.03.022

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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*

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School of Life Science and Biotechnology, Dalian University of Technology, P. R. China

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Zhenzhen Xu and Yafeng Zheng contributed equally to this work.
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Correspondence: Prof. Zhilong Xiu (zhlxiu@dlut.edu.cn), School of Life Science and

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Biotechnology, Dalian University of Technology, No.2 Linggong Road, Ganjingzi District,

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Dalian City, Liaoning Province, P. R. China, 116024.

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Graphical abstract
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Highlights
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 Molasses and CSLP were developed as carbon and nitrogen source, respectively.
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 4.49 g/(L.h), A maximum lactic acid productivity published so far.

 Microbial consortium adapted well with high molasses concentration up to 500 g/L.
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 Economic analysis indicated that lactic acid fermentation cost was 448 USD/ton.
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Abstract
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Sugarcane molasses, a waste from sugar manufacturing processes, has promising

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future to be utilized as a cheap carbon source for lactic acid production. In this study, a

newly microbial consortium, CEE-DL15, for the conversion of sugarcane molasses to lactic

acid was selected and evaluated. The consortium was screened from cattle stomach content

and mainly consisted of Clostridium sensustricto (57.29%), Escherichia (34.22%), and

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Enterococcus (5.32%). Lactic acid production was explored under the deficiency of

sterilization and molasses acidification, with corn steep liquor powder used as organic

nitrogen source. In batch fermentations using sugarcane 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

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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

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and a maximum productivity of 4.49 g/(L.h), which is the best lactic acid productivity from

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molasses published so far. Economic analysis indicated that lactic acid fermentation cost

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was only 448 USD/ton using molasses and corn steep liquor powder, which was only

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37.7% of the total cost when compared with glucose as carbon source and MRS medium as
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nitrogen source. This work demonstrated that the high adaptation to molasses of microbial
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consortium CEE-DL15 might be a promising alternative for the economical production of
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lactic acid.
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Keywords: Microbial consortium, Lactic acid, Sugarcane molasses, Corn steep liquor
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powder
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1. Introduction
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Lactic acid is an important platform chemical that has been applied in foods, cosmetics,

textiles, pharmaceuticals and many other industrial fields. Biotechnological lactic acid
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production has several advantages over chemical approaches regarding environmental

issues, good utilization of diverse substrates, and the feasibility to produce optically pure

lactic acid forms [1]. However, higher production costs have hindered the large-scale

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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

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the economic production of lactic acid. Studies on lactic acid fermentation from raw

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materials as fermentative substrates have suggested numerous candidates, such as

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sugarcane molasses [3, 4], paper sludge [5], cellulose [6], apple pomace [7], kitchen wastes

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[8], Jerusalem artichoke powder [9], etc. As a relatively cheap and abundant raw material,

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sugarcane molasses is a by-product prepared from the liquid waste of sugar production and
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the output of sugarcane molasses in China is about 3 million tons annually, which makes it
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an available raw material for microbial production [10]. Sugarcane molasses contains about
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50% (w/v) total sugar (sucrose, glucose, and fructose), 0.5-0.9% (w/v) nitrogen, 10% (w/v)
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inorganic salts, etc [11, 12]. However, some hazardous substances such as
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5-hydroxymethylfurfural and excessive metallic ions are generated during sugar

manufacturing process, which are toxic to cell growth, causing low conversion yield and
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productivity [13]. To eliminate the inhibition of hazardous substances and improve

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sugarcane molasses utilization, genetic engineering strains or mutagenic strains were

applied to lactic acid production [14, 15]. Escherichia coli WYZ-L, enhancing expression
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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

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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 utilization of cane molasses/glucose carbon sources was

considered to improve the utilization of cane molasses. A titer of 168.3 g/L L-lactic acid

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was obtained by a Bacillus coagulans strain H-1 after 78 h fed-batch fermentation, with a

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productivity of 2.1 g/(L.h) and a yield of 0.88 g/g [4]. Although there were many efforts to

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improve utilization of sugarcane molasses, the productivity was still not too high due to the

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inhibition of the impurities in cane molasses. Cane molasses contains a number of

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inorganic and organic inhibitors, which may impede the metabolism of cells. Therefore,
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cane molasses was usually used in fermentation after pretreatment to reduce the level of
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various inhibitors before application in fermentation. However, pretreatment will raise the
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fermentation cost of cane molasses and increase workload. If no pretreatment of cane

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molasses is allowed, it would be undoubtedly economical for fermentation. Moreover, open

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fermentation is a great advantage for bio-chemicals production while autoclaving would

colorize the broth, lead to sugar losses and increase production cost. If these operation steps
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are reduced, a large amount of energy could be saved for economical lactic acid production
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using raw materials.

Currently, almost all commercial lactic acid production is based on carbohydrate

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fermentations using pure cultures of lactic acid bacteria [16]. In contrast to pure culture

fermentations, microbial consortium offers the advantages of no requirement of

sterilization, versatile fermentation conditions and utilization of complex feedstock. Liang

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et al demonstrated the feasibility of lactic acid production from commercial potato peel

waste by mixed culture fermentations operated in batch and sequencing batch modes [17].

The highest volumetric productivity of lactic acid was achieved in a continuous stirred-tank

reactor (CSTR) by microbial community [16]. Although the final lactic acid concentration

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was not high, these researches implied that microbial consortium might be an alternative to

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pure strains for efficient degradation of complex substrates with multiple impurities, which

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has characteristics of flexible metabolism and low requirement for operation. Up to now,

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microbial consortium is applied to bio-chemicals production, e.g. 1,3-propanediol [18-21]

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and bioenergy, e.g. ethanol [22], butanol [23] and hydrogen [24]. In these studies, microbial
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consortium exhibits advantages such as high adaptation to raw materials, simple
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requirement for nutrients, and unaffected performance under non-sterile conditions.
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The aim of this study was to evaluate a microbial consortium for efficient conversion
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of sugarcane molasses to lactic acid under open operation conditions. Microbial consortium
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CEE-DL15 was isolated, which could convert sugarcane molasses to lactic acid efficiently.

The physiological and biochemical characteristics of CEE-DL15 were determined through

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16S rRNA gene analysis. Then batch fermentation was carried out to evaluate the
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consortium adaptation to cane molasses without sterilization and acidification. Meanwhile,

the microbial community structures of CEE-DL15 during the fermentation process were
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also analyzed and illustrated. Finally, economic evaluation was developed and compared

among different research routes to give insight of lactic acid production.

2. Materials and methods

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2.1. Chemicals

Sugarcane molasses containing a sucrose content of 28.71% (w/v), fructose content of

10.35% (w/v) and glucose content of 3.77% (w/v), was purchase from Liuzhou, China.

Corn steep liquor powder (CSLP) with a nitrogen content of 9% (w/v) was afforded by

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Yuancheng biotechnology company, Liaoning, China. All the medium containing sugarcane

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molasses were freshly prepared and used for fermentation. All other chemicals were of

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analytical grade and commercially available.

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2.2. Medium

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Enrichment medium used for bacterial enrichment contains the following components
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[25]: 40 g/L glucose, 5 g/L yeast extract, 5 g/L sodium fumarate, 0.1 g/L monensin, 3 g/L
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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 enrichment medium was sterilized before incubation.

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Seed medium used for seed culture contains the following components [26]: 30 g/L
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glucose, 2.5 g/L yeast extract, 5 g/L peptone, 5 g/L beef extract, 2 g/L sodium acetate, 2
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g/L ammonium citrate, 0.2 g/L MgSO4, 0.05 g/L MnSO4. The medium was sterilized
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before incubation.

Fermentation medium (MRS medium) used for lactic acid production contained the
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following components [26]: 5 g/L yeast extract, 10 g/L peptone, 10 g/L beef extract, 5 g/L

sodium acetate, 2 g/L K2HPO4, 2 g/L ammonium citrate, 0.58 g/L MgSO4, 0.25 g/L MnSO4,

1 mL tween 80. Sugarcane molasses was used as sole carbon source. MRS medium was

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sterilized or not according to experiment design as shown in Fig. 1.

Simplified MRS (S-MRS) medium contained the following components: 2.5 g/L yeast

extract, 5 g/L peptone, 5 g/L beef extract, 5 g/L sodium acetate, 2 g/L K2HPO4, 2 g/L

ammonium citrate, 0.58 g/L MgSO4, 0.25 g/L MnSO4. Sugarcane molasses was used as

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sole carbon source. The medium was not sterilized before incubation.

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CSLP medium contains 18.5 g/L of CSLP. It was not sterilized before incubation.

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2.3. Enrichment of microorganisms

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Cattle stomach was obtained from Dalian slaughter house and all the samples were

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stored at -20 oC for further studies. Stomach content was prepared by adding 100 g fresh
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content of cattle stomach into 100 mL saline solution, mixing for 2 min with a vortex, and
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then filtering by sterile gauze. The filtrate medium was stomach content suspended matter.
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About 2 g of stomach content or 2 mL stomach content suspended matter was inoculated to

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a 250 mL flask sealed with cotton cap or a 250 mL serum bottle containing 100 mL
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enrichment medium at 37 oC and 200 rpm. The enrichment medium was sterilized before
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incubation. After 12 h incubation, 2 mL of the culture broth was transferred to another flask

or serum bottle with the same enrichment medium and incubated for 12 h. This process was
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repeated ten times.

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2.4. Batch fermentation in 5 L bioreactor

In the experiments of operation conditions comparison, the sugarcane molasses was

pretreated by acid according to the published method [14], while it was used directly

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without pretreatment in the batch and fed-batch fermentation experiments.

The batch fermentation was carried out in a 5 L bioreactor containing 2 L fermentation

medium at 37oC and 200 rpm. The inoculum volume was 5% (v/v).

The pH was controlled at 6.3 by automatic addition of 5 mol/L NaOH for the batch

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fermentation in a 5 L bioreactor. Samples were taken periodically to determine the

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concentration of lactic acid, by-products and reducing sugar. An anaerobic environment in

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the bioreactor was produced by sparging with nitrogen gas at 0.1 vvm for 0.5 h before

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inoculation and 3.5 h after inoculation, respectively.

2.5. Analytical methods

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The biomass concentration was measured at 660 nm with the fermentation medium as
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blank. Samples were pretreated by centrifugation at 9720 × g for 10 min, and the
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supernatants were diluted with deionized water to the desired extent. Glucose, fructose and
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sucrose concentrations were analyzed by high performance liquid chromatography (HPLC)

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equipped with a HypersilNH2-S column with a column temperature of 30 oC. Acetonitrile

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and water (75:25, v/v) was the mobile phase with a flow rate of 0.6 mL/min. Lactic acid

and ethanol concentrations were analyzed by HPLC equipped with an Aminex

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HPX-87Hcolumn with a column temperature of 65 oC. The mobile phase was 5 mM H2SO4
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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 ultraviolet 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

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analyzed by HPLC using a chiral column of Chirex 3126 (D)-penicillami with a PDA

detector at 254 nm, 2 mM CuSO 4 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

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The bacterial community composition of microbial consortium CEE-DL15 was studied

by 16S rRNA gene amplicon high-throughput sequencing. Total DNA of selected microbial

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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.

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16S rRNA gene sequences of the consortia have been submitted to the NCBI Sequence
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Read Archive (SRA) under accession number SRP127781.
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3. Results and discussion

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3.1. Evaluation of microbial consortium

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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
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were chosen to determine their ability to convert glucose to lactic acid. Four enrichment
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and evaluation were investigated and the optimum results are presented in Table 1.

Considering anaerobic characteristic of stomach environment, only anaerobic fermentation

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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 fermentation, respectively. It

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was thus chosen as the microbial consortium for subsequent investigation.

Table 1

3.2. Abundance of microbial consortium CEE-DL15

16S rRNA gene amplicon high-throughput sequencing was performed to investigate

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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

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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

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belonged to Clostridium sensu stricto (57.29%) and Escherichia (34.22%), followed by
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Enterococcus (5.32 %), Anaerobacter (1.94 %), and others (1.23 %).
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Table 2
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3.3. Utilization of sugarcane molasses under different operation conditions by microbial

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consortium CEE-DL15
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Sugarcane molasses contains a number of inorganic and organic inhibitors, which may
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impede the metabolism of cells. Therefore, it is generally used as pretreated to reduce the
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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
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previous study, sugarcane molasses and the fermentation medium was generally autoclaved

which consumed a large amount of energy, colorized the broth, and lead to sugar losses.

The characteristic of open fermentation ability owned by microbial consortium CEE-DL15

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makes the fermentation medium be utilized without autoclaving.

In this study, sugarcane molasses utilization under sterilization and acidification

conditions compared with no sterilization and no acidification was investigated under

different initial sugarcane molasses concentration of 100 g/L, 200 g/L and 250 g/L,

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respectively. Lactic acid concentration, yield and productivity from sugarcane molasses via

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non-sterile and non-pretreated process under 250 g/L of sugarcane molasses was compared

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as Fig. 1 shown. The results showed that the microbial consortium can grow well without

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sterilization and acidification. Lactic acid concentration, yield and productivity were a bit

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high in this condition than in sterilization and acidification, implying that the non-sterile
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and non-pretreated fermentation was feasible for conversion of sugarcane molasses to lactic
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acid. A similar tendency was obtained at 100 g/L and 200 g/L of sugarcane molasses under
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sterilization and acidification conditions, respectively, compared with no sterilization and

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acidification. For example, a titer of 35.48 g/L lactic acid, a yield of 0.71 g/g, and a
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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,
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and a productivity of 1.53 g/(L.h) were obtained under no acidification and sterilization.
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Therefore, to reduce the cost of lactic acid production and workload, sugarcane

molasses was directly utilized without any pretreatment and sterilization during
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fermentation process.

Figure 1

3.4. Selection of economical and simplified fermentation medium

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 Organic nitrogen source containing amino acids, nucleotides, and vitamins are

important and essential for cell growth. Though sugarcane 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

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important for large-scale lactic acid production.

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In this study, three different medium were compared and studied for conversion of

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sugarcane molasses to lactic acid. MRS medium including 5 g/L of yeast extract powder,

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10 g/L of beef extract, 10 g/L of peptone and other salts listed in section 2.2 was selected

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according to a previous report [26]. Simplified MRS medium included 2.5 g/L of yeast
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extract powder, 5 g/L of beef extract, 5 g/L of peptone and other salts. CSLP medium only
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included 18.5 g/L corn steep liquor powder without additional nutrients according to the
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organic nitrogen contents in MRS medium. The results of the influence of three medium
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over the production and productivity of lactic acid are displayed in Fig. 2. Among the three
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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
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72%, respectively). This indicated that CSLP addition enhanced cell growth while
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sugarcane molasses consumption and lactic acid formation rate increased. The primary

costs associated with lactic acid production include the fermentative substrates of nutrients,
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nitrogen sources and sugars required 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

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molasses could be used for the production 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 reduced while CSLP

medium instead of traditional MRS medium.

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Figure 2

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3.5. Sugarcane molasses tolerance of microbial consortium CEE-DL15 in batch

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fermentations

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The capability of sugarcane molasses utilization by microbial consortium CEE-DL15

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was evaluated with different molasses concentrations (250-500 g/L sugarcane molasses).
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As shown in Fig. 3, microbial consortium CEE-DL15 could tolerate high concentration of
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molasses up to 500 g/L. Cell growth was severely inhibited when sugarcane molasses
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concentration was increased to 400 g/L. Sugar consumption and lactic acid formation was
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reduced.
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Figure 3
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When 500 g/L sugarcane molasses was utilized, cell growth was constant for about 120
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h (from 25 h to 145 h). In the end, about 14.41 g/L sucrose was remained and 130.89 g/L
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lactic acid was produced with a productivity of 0.90 g/(L.h) and a yield of 73%. Total

consumption of glucose and fructose was in 27 h and 105 h, respectively (Fig.4). The

results showed that microbial consortium CEE-DL15 had excellent tolerance to sugarcane

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molasses. The final byproduct concentrations of succinic acid, acetic acid, formic acid and

ethanol were 5.38 g/L, 3.75 g/L, 2.15 g/L and 1.75 g/L, respectively.

Figure 4

Five samples for optical purity of lactic acid at different molasses concentrations were

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analyzed by College of Light Industry and Food Science, South China University of

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Technology. The concentration of lactic acid isomers at different sugarcane molasses

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concentrations was shown in Table 3.

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Table 3

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3.6. Fermentation performance of microbial consortium CEE-DL15 and economic analysis
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Lactic acid production from sugarcane molasses and corn steep liquor powder without
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additional nutrients were evaluated under batch fermentation in 5-L bioreactor. Fig. 5
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illustrated the time courses of batch fermentation of 350 g/L of sugarcane molasses. In 350
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g/L of sugarcane molasses batch fermentation, 112.34 g/L of lactic acid (107.40 g/L
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L-lactic acid and 4.94 g/L D-lactic acid) was obtained after 25 h fermentation with a high
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productivity of 4.49 g/(L.h) and a yield of 81%. To our knowledge, this is the highest

productivity ever reported for lactic acid production for sugarcane molasses utilization.
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Figure 5
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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

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were 2.10 g/(L.h), 2.84 g/(L.h) and 3.54 g/(L.h), respectively. A high consumption rate of

reducing sugar was obtained in sugarcane molasses fermentation by microbial 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.

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Figure 6 showed changes in microbial community structures of CEE-DL15 during the

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fermentation process at initial sugarcane molasses concentration of 350 g/L. the microbial

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community was not conservative at the interspecific level during the total cultivation

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conditions. For the preliminary stage, the microbial community structures in sugarcane

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molasses were similar to its cultivation using glucose as carbon source shown in Table 2.
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When fermentation was developed to log phase (8 h), Escherichia became the dominant
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bacteria with a proportion of 79.46%. The proportion of Clostridium sensu stricto dropped
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to 13.92%, while the abundance of Enterococcus increased to 6.1%. At the end of the
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fermentation (24 h), Escherichia and Enterococcus increased continually to 83.3% and
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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

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using sugarcane molasses as carbon source, accounted for 2.21%. As shown in Fig. 5,
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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 abundance changes of microbial community structures might imply
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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.

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 Figure 6

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

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molasses under MRS medium by Lactobacillus delbrueckii. To avoid sugarcane molasses

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inhibition, co-feeding regulation strategy was applied for lactic acid production. The

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co-feeding strategy using cane molasses and glucose will supply sufficient carbon sources

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for lactic acid fermentation without inducing too high concentration of hazardous

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substances. 168.3 g/L L-lactic acid was obtained by a Bacillus coagulans strain H-1 with a
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productivity of 2.1 g/(L.h) and a yield of 88%, using a co-feeding strategy based on the
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utilization of the mixture containing 149 g/L cane molasses and 185 g/L glucose [4].
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Moreover, mutant and genetic strains were evolved and screened out to improve molasses
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utilization and productivity. 75 g/L lactic acid with a yield of 85%, and a productivity of
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1.18 g/(L.h) was produced by E. coli WYZ-L which was an enhanced expression of the

sucrose operon (cscA and cscKB) [15]. High concentration of lactic acid (166 g/L) was
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obtained with a productivity of 4.2 g/(L.h) by Lactobacillus delbrueckii mutant Uc-3 [14].
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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
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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 productivity of 4.49

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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 production includes

fermentative substrates of nutrients, nitrogen sources and carbon sources [2]. In this study,

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the cost of carbon sources and nitrogen sources was analyzed to evaluate the batch

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fermentation for lactic acid production by microbial consortia CEE-DL15. The best

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reported results of lactic acid production from glucose and molasses were selected for

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comparison with our research [14, 29] and the results were presented in Table 4.

Table 4
U 𝐶𝑖 𝑃𝑖
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The cost of carbon and nitrogen sources was defined as (𝑖 = 𝑐𝑠, 𝑛𝑠), where 𝐶𝑐𝑠 ,
𝐶𝐿𝐴
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𝐶𝑛𝑠 and 𝐶𝐿𝐴 represent the concentrations of carbon source, nitrogen sources in medium
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and lactic acid produced, respectively. 𝑃𝑐𝑠 and 𝑃𝑛𝑠 represent the prices of carbon source
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and nitrogen source. Costs and energy consumption were per ton lactic acid produced. The
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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,
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peptone and beef extract were approximatly 5944 USD/ton, 4458 USD/ton and 4458
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USD/ton, respectively. The price of corn steep liquor powder in our study was

approximately 446 USD/ton. Moreover, the cost of sterilization was also analyzed and
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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

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was assigned as 4.2 kJ/(kg.oC), the specific heat of water. Costs and energy consumption in

Table 4 were per ton of required 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

T
[4]. Although the low productivity was obtained in their study, cane molasses used in this

IP
study was not pretreated, and the organic nitrogen in the fermentation medium was only 1

R
g/L corn steep powder. The cost analysis and comparison for carbon source, nitrogen

SC
source and sterilization in Table 4 showed that the cost of cane molasses occupied almost

U
50-60% portion of the cost of glucose in different studies [14, 28, 29]. The cost for MRS
N
medium including yeast extract, peptone and beef extract occupied over 50% portion of
A
total cost for materials and sterilization [28, 29], while the cost of CSLP was only 10%
M

compared to using MRS as nitrogen source. Economic analysis indicated that lactic acid
D

production cost was 1189 USD/ton using glucose as carbon source and MRS medium as
TE

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
EP

mutant Uc-3 in batch fermentation. In our study, lactic acid production cost was only 448
CC

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
A

carbon source and MRS medium as nitrogen source. Therefore, lactic acid production

strategy from sugarcane molasses and CSLP by microbial consortium CEE-DL15 is

preferable on an economical viewpoint.

19
 It should be pointed out that sugarcane molasses should be controlled by acidification

according to the study of Dumbrepatil A et al [14]. Cane molasses contains a number of

inorganic and organic inhibitors, which may impede the metabolism of cells. Therefore,

cane molasses needs to be pretreated to reduce the level of various inhibitors. However,

T
pretreatment will raise the fermentation cost of cane molasses and increase workload. In

IP
the present study, sugarcane molasses was used without any pretreatment, which might be

R
due to the strong tolerance of microbial consortium CEE-DL15. Moreover, the

SC
characteristic of open fermentation ability owned by microbial consortium CEE-DL15

U
makes the fermentation medium be utilized without autoclaving.
N
4. Conclusion
A

In this study, we successfully achieved high production of lactic acid and high
M

tolerance of molasses with a selected microbial consortium CEE-DL15. The evaluation of

D

CEE-DL15 has the potential for cost-effective production of lactic acid using CSLP as
TE

medium without additional nutrients supplements. 112.34 g/L Lactic acid (107.40 g/L
EP

L-lactic acid and 4.94 g/L D-lactic acid), with a yield of 0.81 g/g, a productivity of 4.49

g/(L.h), which is the best lactic acid productivity from molasses published so far was
CC

obtained when 350 g/L sugarcane molasses were utilized without any pretreatment and
A

sterilization. This study provides an efficient and economical option for the utilization of

raw materials in microbial production.

Acknowledgements

20
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

(DUT17ZD209).

References

T
[1] M. Sauer, D. Porro, D. Mattanovich, P. Branduardi, Microbial production of organic acids: expanding the

IP
markets, Trends Biotechnol. 26(2) (2008) 100-108. https://doi.org/10.1016/j.tibtech.2007.11.006.
[2] M.A. Abdel-Rahman, Y. Tashiro, K. Sonomoto, Recent advances in lactic acid production by microbial

R
fermentation processes, Biotechnol. Adv. 31(6) (2013) 877-902.

SC
https://doi.org/10.1016/j.biotechadv.2013.04.002.
[3] B. Flores-Albino, L. Arias, J. Gomez, A. Castillo, M. Gimeno, K. Shirai, Chitin and L(+)-lactic acid
production from crab (Callinectes bellicosus) wastes by fermentation of Lactobacillus sp. B2 using sugar
cane molasses as carbon
https://doi.org/10.1007/s00449-012-0706-4.
source, Bioprocess Biosyst.

U Eng. 35(7) (2012) 1193-1200.

N
[4] K. Xu, P. Xu, Efficient production of L-lactic acid using co-feeding strategy based on cane
molasses/glucose carbon sources, Bioresour. Technol. 153 (2014) 23-29. https://doi.org/
A
10.1016/j.biortech.2013.11.057.
[5] N.K. Budhavaram, Z.L. Fan, Production of lactic acid from paper sludge using a cid-tolerant, thermophilic
M

Bacillus coagulan strains, Bioresour. Technol. 100(23) (2009) 5966-5972.

https://doi.org/10.1016/j.biortech.2009.01.080.
[6] M.S. Ou, L.O. Ingram, K.T. Shanmugam, L: (+)-Lactic acid production from non-food carbohydrates by
D

thermotolerant Bacillus coagulans, J. Ind. Microbiol. Biotechnol. 38(5) (2011) 599-605.

TE

https://doi.org/10.1007/s10295-010-0796-4.
[7] B. Gullon, R. Yanez, J.L. Alonso, J.C. Parajo, L-lactic acid production from apple pomace by sequential
hydrolysis and fermentation, Bioresour. Technol. 99(2) (2008) 308-319.
EP

https://doi.org/10.1016/j.biortech.2006.12.018.
[8] B. Zhang, P.J. He, N.F. Ye, L.M. Shao, Enhanced isomer purity of lactic acid from the non -sterile
fermentation of kitchen wastes, Bioresour. Technol. 99(4) (2008) 855-862.
CC

https://doi.org/10.1016/j.biortech.2007.01.010.
[9] L. Wang, Z. Xue, B. Zhao, B. Yu, P. Xu, Y. Ma, Jerusalem artichoke powder: a useful material in
producing high-optical-purity l-lactate using an efficient sugar-utilizing thermophilic Bacillus coagulans
A

strain, Bioresour. Technol. 130 (2013) 174-180. https://doi.org/10.1016/j.biortech.2012.11.144.

[10] Y. Ni, Y. Wang, Z. Sun, Butanol production from cane molasses by Clostridium saccharobutylicum
DSM 13864: batch and semicontinuous fermentation, Appl. Biochem. Biotechnol. 166(8) (2012)
1896-1907. https://doi.org/10.1007/s12010-012-9614-y.
[11] Kaseno, T. Kokugan, The effect of molasses pretreatment by ceramic microfiltration membrane on
ethanol fermentation, J. Ferment. Bioeng. 83(6) (1997) 577-582.
https://doi.org/10.1016/s0922-338x(97)81140-3.

21
[12] Y.P. Liu, P. Zheng, Z.H. Sun, Y. Ni, J.J. Dong, L.L. Zhu, Economical succinic acid production from
cane molasses by Actinobacillus succinogenes, Bioresour. Technol. 99(6) (2008) 1736-1742.
https://doi.org/10.1016/j.biortech.2007.03.044.
[13] M. Bicker, J. Hirth, H. Vogel, Dehydration of fructose to 5-hydroxymethylfurfural in sub- and
supercritical acetone, Green Chem. 5(2) (2003) 280-284. https://doi.org/10.1039/b211468b.
[14] A. Dumbrepatil, M. Adsul, S. Chaudhari, J. Khire, D. Gokhale, Utilization of molasses sugar for lactic
acid production by Lactobacillus delbrueckii subsp delbrueckii mutant Uc-3 in batch fermentation, Appl.
Environ. Microbiol. 74(1) (2008) 333-335. https://doi.org/10.1128/AEM.01595-07.

T
[15] Y.Z. Wang, K.P. Li, F. Huang, J.H. Wang, J.F. Zhao, X. Zhao, E. Garza, R. Manow, S. Grayburn, S.D.
Zhou, Engineering and adaptive evolution of Escherichia coli W for L-lactic acid fermentation from

IP
molasses and corn steep liquor without additional nutrients, Bioresour. Technol. 148 (2013) 394-400.
https://doi.org/10.1016/j.biortech.2013.08.114.

R
[16] D.H. Kim, M.K. Lee, Y. Hwang, W.T. Im, Y.M. Yun, C. Park, M.S. Kim, Microbial granulation for
lactic acid production, Biotechnol. Bioeng. 113(1) (2016) 101-111. https://doi.org/10.1002/bit.25540.

SC
[17] S.B. Liang, K. Gliniewicz, A.T. Gerritsen, A.G. McDonald, Analysis of microbial community variation
during the mixed culture fermentation of agricultural peel wastes to produce lactic acid,
Bioresour.Technol. 208 (2016) 7-12. https://doi.org/10.1016/j.biortech.2016.02.054.

U
[18] L.L. Jiang, H.F. Liu, Y. Mu, Y.Q. Sun, Z.L. Xiu, High tolerance to glycerol and high production o f
1,3-propanediol in batch fermentations by microbial consortium from marine sludge, Eng. Life Sci. 17(6)
N
(2017) 635-644. https://doi.org/10.1002/elsc.201600215.
A
[19] J.J. Zhou, J.T. Shen, L.L. Jiang, Y.Q. Sun, Y. Mu, Z.L. Xiu, Selection and characterizati on of an
anaerobic microbial consortium with high adaptation to crude glycerol for 1,3-propanediol production,
M

Appl. Microbiol. Biotechnol. 101(15) (2017) 5985-5996. https://doi.org/10.1007/s00253-017-8311-8.

[20] D. Dietz, A.P. Zeng, Efficient production of 1,3-propanediol from fermentation of crude glycerol with
mixed cultures in a simple medium, Bioprocess Biosyst. Eng. 37(2) (2014) 225-233.
D

https://doi.org/10.1007/s00449-013-0989-0.
[21] Y.Q. Sun, J.T. Shen, L. Yan, J.J. Zhou, L.L. Jiang, Y. Chen, J.L. Yuan, E.M. Feng, Z.L. Xiu, Advances
TE

in bioconversion of glycerol to 1,3-propanediol: Prospects and challenges, Process Biochem. 71 (2018)

134-146. https://doi.org/10.1016/j.procbio.2018.05.009.
[22] R. Du, J.B. Yan, S.Z. Li, L. Zhang, S.R. Zhang, J.H. Li, G. Zhao, P.L. Qi, Cellulosic ethanol production
EP

by natural bacterial consortia is enhanced by Pseudoxanthomonas taiwanensis, Biotechnol. Biofuels 8

(2015) 1-10. https://doi.org/10.1186/s13068-014-0186-7.
CC

[23] P.F. Wu, G.Y. Wang, G.H. Wang, B.T. Borresen, H.J. Liu, J.N. Zhang, Butanol production under
microaerobic conditions with a symbiotic system of Clostridium acetobutylicum and Bacillus cereus,
Microb. Cell Fact. 15 (2016) 1-11. https://doi.org/10.1186/s12934-016-0412-z.
[24] S. Sittijunda, A. Reungsang, Biohydrogen production from waste glycerol and sludge by anaerobic
A

mixed cultures, Int. J. Hydrogen Energy 37(18) (2012) 13789-13796.

https://doi.org/10.1016/j.ijhydene.2012.03.126.
[25] R. H. Lin, X. L. Huang, W. Q. Huang, Q. P. Tan, D. Sun, Rapid isolation and identifi cation of a
succinic acid producing strain, Food Sci. Technol. (Chinese) 37(1) (2012) 13-16. https://doi.org/
10.13684/j.cnki.spkj.2012.01.018.
[26] L.F. Coelho, C.J.B. de Lima, M.P. Bernardo, J. Contiero, d(-)-Lactic acid production by Leuconostoc

22
 mesenteroides B512 using different carbon and nitrogen sources, Appl. Biochem. Biotechnol. 164(7)
(2011) 1160-1171. https://doi.org/10.1007/s12010-011-9202-6.
[27]D.M. Liu, H. Wu, Y.G. Yu, X.F. Li, W.J. Guo, W.J. Zeng, Chiral separation and det ermination of L-and
D-lactic acid in pickles by high-performance liquid chromatoraphy, Modern Food Sci. Technol. (in
Chinese) 23(8) (2007) 74-76. https://doi:10.13982/j.mfst.1673-9078.2007.08.024.
[28] B.P. Calabia, Y. Tokiwa, Production of D-lactic acid from sugarcane molasses, sugarcane juice and
sugar beet juice by Lactobacillus delbrueckii, Biotechnol. Lett. 29(9) (2007) 1329-1332.
https://doi.org/10.1007/s10529-007-9408-4.

T
[29] S.K. Moon, Y.J. Wee, G.W. Choi, A novel lactic acid bacterium for the produ ction of high purity
L-lactic acid, Lactobacillus paracasei subsp paracasei CHB2121, J. Biosci. Bioeng. 114(2) (2012)

IP
155-159. https://doi.org/10.1016/j.jbiosc.2012.03.016.

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A

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A
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Fig. 1. Lactic acid production under different operation conditions at initial sugarcane

molasses concentration of 250 g/L. Fermentation was carried out at 37 oC in 250 mL serum
D

bottle containing 100 mL medium under anaerobic conditions. MRS medium was selected
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and fermentation was complete at 48 h. Values are means of three independent

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fermentations.
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A

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

at initial sugarcane molasses concentration of 250 g/L. Fermentation was carried out at
D

37oC and 200 rpm in 250 mL serum bottle containing 100 mL medium under anaerobic
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conditions. MRS, S-MRS and CSLP medium were selected. The fermentation for MRS,
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S-MRS and CSLP medium was complete at 48 h, 42 h, and 36 h, respectively. Values are

means of three independent fermentations.

CC
A

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 T
R IP
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A
M

Fig. 3. The tolerance of microbial consortium CEE-DL15 to sugarcane molasses in batch

fermentations in 5 L bioreactor. CSLP medium was selected. Fermentation was carried out
D

at 37oC, pH 6.3 and 200 rpm. CSLP medium was selected without sterilization. (A) Lactic
TE

acid concentration. (B) Microbial growth. (C) Fermented sugar concentration.

EP
CC
A

26
 T
R IP
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A
M
D
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EP
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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

A

out at 37oC, pH 6.3 and 200 rpm. CSLP medium was selected without sterilization. (A)

Fermented sugar and lactic acid concentration. (B) Sucrose, fructose and glucose

concentration. (C) By-products concentration.

27
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R IP
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U
N
A
M
D
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EP
CC

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

A

out at 37oC, pH 6.3 and 200 rpm. CSLP medium was selected without sterilization. (A)

Fermented sugar and lactic acid concentration. (B) Sucrose, fructose and glucose

concentration. (C) By-products concentration.

28
 Escherichia
Clostridium sensu
stricto
90 Enterococcus
Anaerobacter
Melissococcus

T
Methanobrevibacter
Pseudocitrobacter
Lactobacillus

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Enterobacter
Pluralibacter
Other
60
Percentage (%)

R
SC
30

U
N
0
A
Inoculum 8h 24 h
______________________
Batch fermentation
M

Fig. 6. The bacterial community structure of CEE-DL15 during the fermentation at initial
D

sugarcane molasses concentration of 350 g/L in 5 L bioreactor.

TE
EP
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A

29
Table 1

Enrichment and evaluation of microbial consortium for the coversion of glucose to lactic

acid.

T
IP
Lactic acid Yield
Origin Enrichment Fermentation

R
(g/L) (g/g)

SC
1 Stomach content suspended matter anaerobic anaerobic 40.95 0.88

2 Stomach content suspended matter aerobic

U
anaerobic 35.67 0.77
N
3 Stomach content anaerobic anaerobic 41.80 0.90
A
4 Stomach content aerobic anaerobic 29.76 0.65
M
D
TE
EP
CC
A

30
Table 2

Microbial community analysis of the original consortium of CEE-DL15

Taxonomy Percentage (%)

T
Clostridium sensu stricto 57.29

IP
Escherichia 34.22

R
Enterococcus 5.32

SC
Anaerobacter 1.94

Melissococcus 0.18

Exiguobacterium
U 0.14
N
Unclassified 0.91
A
M
D
TE
EP
CC
A

31
 Table 3

The concentration of lactic acid isomers at different sugarcane molasses concentrations.

Sugarcane
Fermentation Total lactic L-lactic acid D-lactic acid L-lactic acid

T
Samples molasses
time (h) acid (g/L) (g/L) (g/L) （%）
（g/L）

IP
1 250 5 28.66 25.11 3.65 87.61
2 250 19 85.95 78.42 7.53 91.24

R
3 350 25 112.34 107.4 4.94 95.60

SC
4 400 40 36.52 31.26 5.26 85.60
5 500 40 28.32 23.85 4.47 84.22

U
N
A
M
D
TE
EP
CC
A

32
 R I
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Table 4

U
N
Costs analysis and comparison for carbon source, nitrogen source and sterilization.*

A
*Costs and energy consumption were per ton lactic acid produced.

M
References D Concentration
Lactic acid

Yield Productivity
Carbon source Nitrogen source
Sterilization

Energy consumption C
TE
(US dollar $) (US dollar $)
(g/L) (g/g) (g/(L.h)) (106 kJ) (US d
yeast extract (5 g/L),
EP

[29] 192.0 0.96 3.99 Glucose 521 peptone (10 g/L), 619 1.97 49
beef extract (10 g/L)
CC

[14] 166.0 0.95 4.20 Molasses 263 yeast extract (5 g/L) 179 2.28 57

165.7 0.92 1.60 Glucose 546 CSLP (1 g/L) 3 2.28 57

[4]
A

Glucose/
168.3 0.88 2.10 446 CSLP (1 g/L) 3 2.25 56
Molasses

[15] 75.0 0.85 1.18 Molasses 294 CSLP -a 5.04 12

yeast extract (5 g/L),

[28] 107.0 0.90 1.48 Molasses 278 619 3.53 88
peptone (10 g/L),

33
 R I
SC
beef extract (10 g/L)

U
This study 112.3 0.81 4.49 Molasses 375 CSLP (18.5 g/L) 73 0 0

N
Costs and energy consumption were per ton lactic acid produced.

A
a
The mass of CSLP was not given.
b
The cost of nitrogen source was not included.

M
D
TE
EP
CC
A

34