SCIENTIFIC PAPER

PRODUCTION OF LACTIC ACID BY Enterococcus faecalis ON WASTE

GLYCEROL FROM BIODIESEL PRODUCTION

Jovan Ćirić1*, Nataša Joković2, Slavica Ilić1, Sandra Konstantinović1, Dragiša Savić1,

Vlada Veljković1

1
Faculty of Technology, University of Niš, Bulevar Oslobođenja 124, 16000 Leskovac, Serbia
2
Faculty of Sciences and Mathematics, University of Niš, Višegradska 33, 18000 Niš, Serbia
*
Present address: Research and Development Center “ALFATEC”, Bulevar Nikole Tesle, 18000 Niš, Serbia

Received 10.10.2019.

Accepted 12.10.2019.

*
Corresponding author: joca.ciric@gmail.com (J. Ćirić) +381 63 1059371

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Abstract

Waste glycerol from biodiesel production is a valuable raw material that has been used
to produce valuable microbial metabolites. In this work, the possibility of microbial utilization
of waste glycerol obtained as a by-product in biodiesel production from sunflower and
rapeseed oil by the lactic acid bacterium Enterococcus faecalis MK3-10A on laboratory level
was studied. For comparison, pure glycerol and glucose were used as carbon sources. The
kinetics of the microbial biomass growth, the carbon source utilization, and the lactic acid
production were monitored. The bacterium E. faecalis MK3-10A better grew in the media
with glucose or pure glycerol as a carbon source but the lactic acid production rate was the
highest (14.6 mg/ml/day) in the medium with waste glycerol from the sunflower oil-based
biodiesel production. Therefore, this waste glycerol might be a promising carbon source for
lactic acid-bacteria cultivation and lactic acid production.

Keywords: Enterococcus faecalis, waste glycerol, biodiesel, lactic acid

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Introduction

With a significant increase in industrial biodiesel production worldwide, a huge

amount of waste glycerol has been created, which lowers the pure and waste glycerol prices
on the world market [1]. The demand and the glycerol price can significantly increase if new
possibilities of its microbiological conversion and use could be applied in industrial
fermentations. Many microorganisms, such as Escherichia coli, bacteria of genus Klebsiella,
Enterobacter, Gluconobacter and Clostridium, fungi of genus Candida and Aspergillus and
others, have been reported to successfully convert glycerol into valuable products, such as
1,3-propanediol, n-butanol, ethanol, lactic acid, citric acid and others [2-6]. Thereby, glycerol
has the potential to become one of the cheapest natural substrates to produce valuable
industrial metabolites.
Several studies about microbiological conversion of waste glycerol to lactic acid using
various strains, such as Lactobacillus casei 2125, Lactobacillus delbrueckii 2025,
Lactobacillus pentosus 2912, Bacillus laevolacticus 2464 NCIM, Paenibacillus pabuli 2912,
Lactococcus lactis 3041 MTCC, Enterococcus faecalis W11, Bacillus sp. (KF781350),
Enterococcus faecalis QU 11 and Rhizopus oryzae NRRL 395 have been published [7-11].
Vodnar et al. [11] have studied the possibility of the lactic acid production by mold Rhizopus
oryzae NRRL 395 on waste glycerol obtained from a soybean oil-based biodiesel production.
It is known that some strains of Enterococcus faecalis can produce lactic acid on glycerol [12-
15] or waste glycerol from biodiesel production [8, 10, 11]. Doi [8] and Murakami et al. [10]
have cultivated the metabolically-engineered E. faecalis strains in the media with waste
glycerol in order to study the possibility of its application. They demonstrated that the
metabolically-engineered E. faecalis can efficiently convert crude glycerol to lactic acid. Doi
[8] also reported that the microbial growth decreased under anaerobic conditions when the E.
faecalis W11 was cultivated at higher glycerol concentrations; however, in aerobic conditions,
the metabolic activities were significantly better. Further research with E. faecalis W11 under
aerobic conditions indicated that the lactic acid fermentation was the main aerobic metabolic
pathway utilizing glycerol at a high concentration and resulted in the higher produced
amounts than those achieved in the previous studies, which was very specific and
characterized as a phenomenon [16]. A few studies reported a stimulating effect of the
combined carbon sources on the primary and secondary microbial metabolisms [17, 18].

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 The aim of this work was to study the possibility of lactic acid production by the wild
bacterium E. faecalis MK3-10A, isolated from a traditional dairy product, cultivated on the
medium containing waste glycerol as the carbon source. In order to compare efficiencies of
the processes, the bacteria were simultaneously cultivated under the same experimental
conditions on pure glycerol and glucose as carbon sources.
Experimental

Microorganism
The used strain MK3-10A was isolated from a seven-month matured milk cream
(kaymak) sample [19] and identified as E. faecalis by Joković et al. [20]. A culture grown 48
h at 28 ºC on the MRS broth was used as an inoculum in the amount of 2% of the total
volume.
Fermentation conditions
The bacterium were cultivated in 500 ml flasks filled with 300 ml of the MRS (de
Man, Rogosa, Sharpe) carbon source free broth (10 g/l peptone; 10 g/l meat extract; 5 g/l
yeast extract; 20 g/l carbon source; 2 g/l K2HPO4; 5 g/l CH3COONa; 2,5 g/l MgSO4 x 7 H2O;
0,2 g/l MnSO4 x 7 H2O; pH 6,4) at 28 ºC. The sources of carbon were added afterward in the
carbohydrate-free media. All compounds were pharmaceutical purity (Sigma Aldrich).
Glucose, pure glycerol, waste glycerol from sunflower and rapeseed oil-based biodiesel
productions and a mixture of glycerol and glucose were used as carbon sources; the used
concentrations of the carbon sources are given in Table 1. During the fermentation, 10 ml of
the fermentation broth was sampled every 2 h under aseptic conditions for further analyses.
All experiments were carried out in triplicate.
Table 1
Analytical methods

Microbial growth was monitored spectrophotometrically at λ = 620 nm, using a Cole

Parmer 2100 UV/VIS spectrophotometer during the fermentation.
The concentrations of glycerol, glucose and lactic acid were determined using an
HPLC method. The samples of the fermentation medium (10 ml) were centrifuged at 3000
min-1 for 20 min and the supernatant was filtrated through a microfilter (0.45 µm). The
Agilent 1100 Series chromatograph with an Aminex HPX-87H column was used for the
separation of the components. After the application of 20 µl of the sample, elution was carried
out with 5 mM H2SO4 (Sigma Aldrich HPLC purity Sulfuric acid) at 50 ºC and the flow rate
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of 0.6 ml/min. Glucose and glycerol were detected by an IR detector and their concentrations
were calculated on the base on the retention time and peaks of pure (p.a.) glucose and
glycerol.
Calculations and statistical analysis
Data for cell growth, glucose and glycerol consumption, and product concentration
were used to calculate consumption rates of glucose Δgluc and glycerol Δglyc (mg/ml/d) and
production rate yield YP/SLA (mg/ml/d) [21]. The percentile of consumption was calculated in
the relation between the decrease of carbon source (spent) with the initial concentration.
Pearson’s correlation coefficient was calculated for all data related to the production
of lactic acid in all experiments using Excel (Microsoft Office 365).
Results and discussion

Variations of biomass, glucose, glycerol, and lactic acid concentrations during the
lactic acid fermentation by E. faecalis MK3-10A in the MRS media with glucose and glycerol
are shown in Figure 1 while the microbial growth change, the consumption rates and relative
consumptions of the carbon sources, as well as the achieved maximum lactic acid
concentration and production rate, are presented in Table 2.
Figure 1
Table 2
Good growth of E. faecalis MK3-10A can be observed in all the used media (Figure
1a). The maximum biomass concentration was achieved in the medium with glucose while it
was slightly less in the PG and WGSO media. In the G-WGRO medium with a mixture of
glucose and WGRO, the growth was reduced compared to the other used carbon sources. The
sustained growth phase (lag phase) lasted for 2 h in the G-PG medium, 4 h in the G-WGSO
and 6 h in the G-WGRO medium, followed by an exponential growth phase of up to 20 h of
fermentation, when the culture entered the stationary phase (Figure 1a).
The increase of the biomass resulted in the consumption of glucose (Figure 1b) in the
first 20 h. During the fermentation process on the G medium, the specific glucose
consumption rate was the highest (12 mg/ml/day) when 61% of the total available amount
was consumed (Table 2). Compared to the G medium, the G-PG and G-WGSO media
provided a smaller specific glucose consumption rate by 6% and 4% respectively. Although
the microbial growth was lower in the G-WGSO medium than in the G medium, the glucose
consumption rate was higher by 8%, which indicated a stimulating effect of waste glycerol on

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glucose metabolism. The lowest growth and glucose consumption rate (46%) were achieved
in the G-WGRO medium, which was ascribed to the negative impact of the impurities
contained in waste glycerol obtained from rapeseed oil.
The glycerol concentration in the G-PG, G-WGSO and G-WGRO media decreased
from the initial 5 g/l by 35%, 31%, and 26%, respectively to reach the consumption rate of 2.4
mg/ml/day, 1.4 mg/ml/day and 1.2 mg/ml/day (Table 2). Based on glucose and glycerol
consumptions, it was concluded that the growth of E. faecalis MK3-10A was a consequence
of glucose utilization as a more accessible carbon source.
Independently of the medium composition, the lactic acid production followed the
growth and consumption of the carbon source and increased until reaching the stagnation
period after 22 h (Figure 1d). The maximum lactic acid concentration (16 mg/ml) was
achieved in the G medium while the maximum concentrations achieved in the G-WGSO and
G-WGRO media were lower by 7% and 10% respectively. The addition of waste glycerol in
the medium with glucose reduced the production of lactic acid.
The maximum lactic acid concentration of 15.8 mg/ml obtained in the WGSO medium
was slightly lower than that observed in the basal G medium (16 mg/ml) but it was higher
than that achieved in the PG and WGRO media (14.6 mg/ml and 9.3 mg/ml, respectively).
The lactic acid production rate in the WGSO was the same as in the basal G medium and 9%
and 78% higher compared to the PG and WGRO media, respectively.
In conclusion, the bacterium E. faecalis did not use all amounts of the available
glucose and glycerol while the produced lactic acid deaccelerated the bacterial growth after
20–22 h of the bioprocess. It is known that lactic acid in the concentration of 1–2% can inhibit
further microbial growth [22-24].
Figure 2 shows the bacterial growth, the substrate consumption, and the lactic acid
production during the E. faecallis MK3-10A cultivation on pure and waste glycerol as a sole
carbon source. From Figure 2a, which shows the change of the microbial growth over time, it
is observed that the best growth (ODmax = 0.64) is achieved in the WGSO medium.
Also, the highest glycerol consumption rate of 7.0 mg/ml/d was achieved in the
WGSO medium while it was 6.5 mg/ml/d in the PG medium (Table 2). The lowest glycerol
consumption rate (3.6 mg/ml/d) and glycerol consumption (21%) was observed in the WGRO
medium (Table 2), which could be explained by the adverse and inhibitory effects of
impurities contained in this type of waste glycerol.

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 The maximum concentration of the produced lactic acid (15.8 mg/ml) was achieved in
the WGSO medium while a slightly lower value of the maximum lactic acid concentration
(14.6 mg/ml) was obtained in the PG medium. As a result of the lower growth, E. faecalis
produced lactic acid at the lowest concentration (9.3 mg/ml) when growing in the WGRO
medium (Figure 2c). The production of lactic acid in the WGRO medium was the lowest
compared to all the studied media and the maximum reached concentration was 9.3 mg/ml.
The analysis of the values of the Pearson’s coefficient showed that the lactic acid
production on the glucose as a sole carbon source (G) was positively and significantly
correlated with the experiments where a part of glucose was substituted with pure glycerol
(G-PG, r = 0.969), glycerol from sunflower- (G-WGSO, r = 0.968) or rapeseed-based
biodiesel production oil (G-WGRO, r = 0.953). Moreover, high values of the correlation were
also calculated by comparing the lactic acid production on glucose with those on pure
glycerol (PG, r=0.994), glycerol from sunflower- (WGSO, r = 0.970) or rapeseed-based
biodiesel production oil (WGRO, r = 0.930) as sole carbon sources.
Figure 2
There are not published results based on the usage of waste glycerol from rapeseed oil
in microbial conversions and lactic acid production, making difficult a relevant comparison.
However, bioconversion of soybean-based waste glycerol to lactic acid has been frequently
studied using various microorganisms [7, 11, 13, 25] but not Enterococcus faecalis. Hong et
al. [13] reported that the E. coli AC-521 strain isolated from soil samples showed a very high
lactic acid production potency on the soybean-based glycerol media, with a final lactic acid
concentration of 85.8 g/L and a molar yield of 0.90. Mazumdar et al. [25] have achieved 50
g/L of L-lactate from 56 g/L of crude glycerol using the E. coli LA20 engineered strain with a
yield (0.93) close to the theoretical maximum (0.967). Also, another metabolically-engineered
strain E. coli B0013-070 converted 20 g/L of crude glycerol to 13.6 g/L of D-lactate with a
yield of 0.67 g/g crude glycerol [26], which is the closest to our conversion results.
The achieved maximum lactic acid production rate and glycerol consumption rate of
E. faecalis MK3-10A in the glucose and glycerol-based media were significantly lower in
comparison with the published results [8] obtained with some modified strains of E. faecalis
grown on waste glycerol from the biodiesel production. Doi [8] reported the maximum lactic
acid concentration of 45–55 mg/ml in the fed-batch conditions in the medium with 30 g/l of
waste glycerol, which was twice higher than the initial glycerol concentration applied in the
present study. Murakami et al. [10] reported that the growth (OD620) of a genetically modified
7
strain E. faecalis in the fed-batch conditions was 44% higher than the growth of E. faecalis
MK3-10A. The former strain consumed all available glycerol within 192 h. In the batch
conditions, strain QU 11 consumed 70–93% from the initial 20 g/l of glycerol and produced
5-15mg/ml of lactic acid. Some researchers have reported that the modified strains of E. coli,
K. pneumoniae and R. oryzae, grown in the media with glycerol, can produce lactic acid in the
concentration higher than 30 mg/ml [11-15]. For instance, with R. oryzae NRRL 395, Vodnar
et al. [11] achieved the highest lactic acid yield (3.7 g/g glycerol, i.e. 48 mg/ml) and the
biomass yield of 0.7 g/g glycerol in the medium containing waste glycerol obtained from the
soybean oil-based biodiesel production in the initial concentration of 75 g/l.
Conclusion

The bacterium E. faecalis MK3-10A showed the best growth in the media containing
either glucose or a mixture of pure glycerol and glucose as a carbon source. Regardless of the
lower growth, the highest lactic acid production (14.6 mg/ml/day) was achieved by cultivating
this strain in the medium containing waste glycerol from the sunflower oil-based biodiesel
production as the sole carbon source. Therefore, this by-product can be considered as a
promising cheap carbon source for further research on the possibility of the lactic acid
bacteria cultivation on an advanced level for lactic acid production.

Acknowledgments
This work has been funded by the Ministry of Education, Science and Technological
Development of the Republic of Serbia (Project III 45001).

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Figure Captions:

Figure 1 Changes of the optical density (biomass) (a), as well as the concentrations of

glucose (b), glycerol (c) and lactic acid (d) during the fermentation of E. faecallis MK3-10A

in the G (♦), G-PG (■), G-WGSO (▲) and G-WGRO (●) media

Figure 2 Changes of the optical density (biomass) (a), as well as the concentrations of

glycerol (b) and lactic acid (c) during the fermentation of E. faecalis MK3-10A in the PG (♦)

WGSO (■) and WGRO medium (▲)

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Table 1 The concentrations of the carbon sources in the MRS medium used for cultivation of

the bacterium Enetococcus faecalis MK3-10A.

Growth medium Medium

Carbon source (g/l)
short
designation Glycerol
Glucose
Pure WGSOa WGROb
Glucose G 20 - - -
Pure glycerol PG - 20 -
Glucose and pure glycerol G-PG 15 5 - -
Waste glycerol from sunflower oil WGSO - - 20 -
Waste glycerol from rapeseed oil WGRO - - - 20
Glucose and waste glycerol from sunflower oil G-WGSO 15 - 5 -
Glucose and waste glycerol from rapeseed oil G-WGRO 15 - 5
a
WGSO - Waste glycerol obtained from the sunflower oil-based biodiesel production.
b
WGRO - Waste glycerol obtained from the rapeseed oil-based biodiesel production.

13
Table 2 The effect of pure and waste glycerol on the microbial growth (ΔOD620max), the

consumption rates of glucose (Δgluc) and glycerol (Δglyc), and the relative consumption carbon

sources (%), as well as the achieved maximum lactic acid concentration (CmaxLA) and

production rate yield (YP/SLA)

ΔOD620max Δgluc Δglyc CmaxLA YP/SLA

Growth media
- (mg/ml/d) (%) (mg/ml/d) (%) mg/ml (mg/ml/d)
G 0.75 12.0 61 - - 16.0 14.6
PG 0.62 - - 6.5 34 14.6 13.4
G-PG 0.69 8.2 55 2.4 35 15.1 13.9
WGSO 0.64 - - 7.0 37 15.8 14.6
WGRO 0.40 - - 3.6 21 9.3 8.2
G-WGSO 0.64 8.9 57 1.4 31 14.8 13.3
G-WGRO 0.48 7.0 46 1.2 26 11.8 10.4

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Figure 1

15
Figure 2

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