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Biochemical Engineering Journal 98 (2015) 3846

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Biochemical Engineering Journal


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Effect of different types of calcium carbonate on the lactic acid


fermentation performance of Lactobacillus lactis
Peng-Bo Yang a,b , Yuan Tian a , Qian Wang a , Wei Cong a,
a
b

National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, PR China
University of Chinese Academy of Sciences, Beijing 100049, PR China

a r t i c l e

i n f o

Article history:
Received 16 November 2014
Received in revised form 4 January 2015
Accepted 18 February 2015
Available online 20 February 2015
Keywords:
Lactic acid
Fermentation
Recycled calcium carbonate
Direct interaction
Afnity
Adsorption

a b s t r a c t
The effect of two different types of calcium carbonate on lactic acid fermentation was investigated in this
study. The results showed that the fermentation performance of calcium carbonate greatly improved
when the recycled particle was used instead of the original particle. The improved fermentation was
evidenced by the pH of the broth, the enhanced production of lactic acid (increase of 33.17%) and the
enhanced cell dry weight (CDW, increase of 19.24%). Analysis of the composition and structure of calcium carbonate revealed that the recycled compound possesses several unique features that allow it to
maintain a higher pH and provide nitrogen during fermentation. The experimental results indicated that
a higher pH value and the addition of nitrogen could increase the fermentation performance, but these
factors were not sufcient to explain the original ndings. Macroscopic and microscopic studies conrmed that direct interactions, such as absorption and particle entrance, occurred between Lactobacillus
lactis-11 and the recycled calcium carbonate particles. These direct interactions may provide a favorable
microenvironment to the cell.
2015 Elsevier B.V. All rights reserved.

1. Introduction
Lactic acid is an organic acid that is used in numerous elds,
such as food preservation, pharmaceuticals, and leather and textile manufacturing [1]. Fermentation has recently been considered
the main method for producing lactic acid [2,3]. During lactic acid
fermentation, the pH value of the broth decreases as the concentration of undissociated lactic acid increases. This condition inhibits
the ability of microbes to produce lactic acid due to the feedback
inhibition effect. Consequently, to obtain a higher production rate
of lactic acid, a neutralizer should be added to the fermentation
broth to stabilize its pH value [4].
According to previous studies, calcium carbonate, calcium
hydroxide, ammonia, sodium hydroxide or potassium hydroxide
could be used as neutralizing agents in the lactic acid fermentation process [5,6]. Ammonia, sodium hydroxide and potassium
hydroxide are rapid and effective neutralizers and can therefore
stabilize the pH value of the broth without producing calcium
sulfate as a byproduct. However, the nal acid and cell concentrations are inhibited due to the toxicity of ammonia and

Corresponding author at: 1 North 2nd Street, Zhongguancun, Haidian District,


Beijing, PR China. Tel.: +86 10 82627060; fax: +86 10 82627066.
E-mail address: weicong@ipe.ac.cn (W. Cong).
http://dx.doi.org/10.1016/j.bej.2015.02.023
1369-703X/ 2015 Elsevier B.V. All rights reserved.

lactic acid toward microbial cells. Calcium alkali is a temperate


neutralizer for lactic acid bacteria and yields a high lactic acid
concentration when added to the broth [7]. However, solid waste
(calcium sulfate) is unavoidably produced in the extraction process.
To solve this problem, a new technology for the production
of lactic acid was developed using calcium carbonate as a neutralizer without yielding calcium sulfate as a byproduct [8]. The
core step of this technology was a displacement reaction that can
convert calcium lactate into soluble lactate and calcium carbonate (recycled calcium carbonate). To ensure that the fermentation
process was environmentally friendly and economical, the recycled calcium carbonate can be re-used as the neutralizer in the
next fermentation batch. An interesting nding from the experiments was that lactic acid bacteria appeared to be more interested
in the recycled calcium carbonate compared with the original calcium carbonate. However, the mechanism for this phenomenon is
unknown.
This study provides the rst demonstration of the effects of
two types of calcium carbonate on lactic acid fermentation. The
constitution, surface morphology and particle size were compared between the calcium carbonate types, and the relationship
between the molecular characteristics and experimental results is
discussed.

P.-B. Yang et al. / Biochemical Engineering Journal 98 (2015) 3846

39

Table 1
Lactic acid fermentation with different conditions (CaCO3 , nitrogen source and pH) by Lactobacillus lactis-11 (Fermentation condition: 5-L stirred fermentor, agitation speed
of 100 rpm, 42 1 C, feeding glucose solution, original calcium carbonate as neutralizing agent).
Conditions

Time (h)

Cell dry weight


(g L1 )

Lactic acid
(g L1 )

Yield
(g lactic acid g1
glucose)

Productivity after
12 h
(g L1 h1 )

Recycled CaCO3
Original CaCO3
Original CaCO3 and pH adjustment
Original CaCO3 and adding N
Recycled CaCO3 and reducing N
Original CaCO3 , pH adjustment and adding N

60
60
60
60
60
60

6.414
5.379
5.766
5.426
6.306
5.878

143
108
124
114
140
130

0.978
0.987
0.982
0.985
0.979
0.980

2.73
2.05
2.38
2.17
2.69
2.50

2. Methods
2.1. Microorganism and media
Lactobacillus lactis-11 (provided by Shandong University, China),
a lactic acid-producing strain of the bacteria, was grown on de Man
Rogosa Sharpe (MRS) [9] medium. The seed culture was grown
in MRS broth at 42 C with shaking at 100 rpm for 12 h, and the
inoculation size was 10% (v v1 ) in all of the experiments. The fermentation broth consisted of (per liter of distilled water) 5 g of
yeast, 10 g of peptone, 10 g of beef extract, 10 g of NaCl, 5 g of sodium
acetate, 2 g of triammonium citrate, 0.4 g of MgSO4 , 0.01 g of MnSO4
and 20 g of glucose.

2.2. Batch fermentation


The batch cultures were performed in a 5-L stirred fermentor
(BaoXing, Shanghai, China). The volume of initial medium was 3.0 L.
No air was introduced to the cultures, and an agitation speed of
100 rpm was employed to ensure the homogeneity of the fermentation broth. The culture temperature was set to 42 1 C. The carbon
source (800 g L1 glucose solution) was fed into the fermentor during the fermentation, and the feeding speed was set according to
the concentration of residual sugar in broth. The neutralizing agent
was calcium carbonate, which was added to the broth before fermentation. The calcium carbonate addition in one batch was 240 g,
and the ratio of glucose to calcium carbonate was about 3:2 (w/w).

2.3. Preparation of recycled calcium carbonate


The recycled calcium carbonate was obtained from the displacement reaction between calcium lactate in the broth and ammonium
carbonate. First, the freshly fermented broth was centrifuged at
4000 g for 25 min, and the supernatant was then poured into
an airtight agitation tank. Afterward, ammonium carbonate was
supplied to the supernatant at a ratio of 1:1 (mol mol1 ), and the
supernatant was agitated at 200 rpm and room temperature for 3 h.
Finally, an additional centrifugation was conducted at 4000 g for
5 min, and the residue obtained was used as the neutralizing agent
in this study.

2.4. Analysis
2.4.1. Determination of the microbe and substance
concentrations
The biomass concentration was expressed as the cell dry weight,
which was determined by measuring the optical density (OD) of the
broth at 620 nm. The calcium carbonate particle should react after
the addition of diluted hydrochloric acid. The optical density was
proportional to the cell dry weight, and one OD unit corresponds
to 0.516 g L1 of biomass.

The glucose concentration was determined using a SBA-40C


biosensor analyzer (Institute of biology, Shangdong Province
Academy of Sciences, P.R. China).
Lactic acid was measured by high-performance liquid chromatography (HPLC) [5].
The nitrogen content was measured using a Flash EA 1112 elemental analyzer (Thermo Fisher Scientic Inc., USA). The sample of
calcium carbonate was dissolved in dilute hydrochloric acid. Each
sample measurement was performed in duplicate, and the average
value is reported. The difference between the values was always
less than 5%.
The content of amino nitrogen was measured by the Coomassie
brilliant blue method.
2.4.2. Electron microscopy and surface elemental analysis
Images of the calcium carbonate particles were taken at
10 kV with a scanning electron microscope (SEM; S-4800, Hitachi
High-Technologies Corporation, Japan). Elemental analyses were
performed using an X-ray energy dispersive spectrometer (EDS)
(EDAX9100, JEOL Ltd., Japan).
2.4.3. Particle size determination
The particle size distribution was analyzed using a Malvin Hydro
2000 Mu laser particle size analyzer. The samples were dispersed in
water by ultrasonic treatment for 2 min before the measurements.
2.4.4. Specic surface area determination
The specic surface area was determined using N2 with a specic surface area analyzer (Autosorb-1, Quantachrome, USA).
3. Results and discussion
3.1. Different fermentation performances obtained using recycled
and original calcium carbonate
Batch fermentations were performed under the same conditions
with the exception of the neutralizing agents. In the different batch
fermentations, recycled and original calcium carbonate were used
as neutralizing agents. As shown in Fig. 1A and B, the use of recycled
calcium carbonate as the neutralizing agent increased the concentration of lactic acid from 108 g L1 to 143 g L1 over the same time
period and under the same fermentation conditions as the experiment with original calcium carbonate. The presence of recycled
calcium carbonate was favorable for the growth of L. lactis-11, as
evidenced by an increase in the cell dry weight from 5.379 g L1 to
6.414 g L1 (see Table 1). The ability of recycled calcium carbonate
to adjust the pH of the solution was better than that of original
calcium carbonate.
The data from the lactic acid accumulation stage (after 12 h)
were selected for further analysis. As shown in Table 1, the production rate of lactic acid obtained using recycled calcium carbonate
as the neutralizing agent reached 2.73 g L1 h1 , which was 33.17%

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P.-B. Yang et al. / Biochemical Engineering Journal 98 (2015) 3846

Fig. 1. Time courses of lactic acid fermentation by Lactobacillus lactis-11: [A, recycled calcium carbonate; B, original calcium carbonate; C, original calcium carbonate and
adjusting pH by the addition of calcium hydroxide solution; D, original calcium carbonate and adding more nitrogen; E, recycled calcium carbonate and reducing part of
nitrogen; F, original calcium carbonate, adjusting pH by the addition of calcium hydroxide solution and adding more nitrogen. , lactic acid; , glucose; , pH; , cell dry
weight of suspension (CDW1 ); , cell dry weight of supernatant (CDW2 )].

higher than the production rate of lactic acid obtained with original
calcium carbonate (2.05 g L1 h1 ).
This result is due to the type of calcium carbonate used. Consequently, the difference between these two types of calcium
carbonate should be further investigated.
3.2. Structural features of recycled and original calcium
carbonate
The composition, particle size and specic surface area of recycled and original calcium carbonate are compared. The results

showed that recycled calcium carbonate produced nitrogen during


fermentation and had a smaller particle size and a higher specic surface area compared with the original calcium carbonate.
The total nitrogen and amino nitrogen contents in recycled calcium carbonate were found to be 0.08% and 0.03%, respectively.
In contrast, the nitrogen content in the original calcium carbonate was below the detection limit (less than 0.01%). The particle
sizes of recycled and original calcium carbonate were 4.682 m
and 12.431 m, respectively, and the specic surface areas of recycled and original calcium carbonate were found to be 8.190 m2 g1
and 0.5615 m2 g1 , respectively.

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41

Fig. 2. SEM images of recycled calcium carbonate at different times during the process of fermentation (A, 0 h; B, 12 h; C, 27 h; D, 39 h; E, 51 h; F, 60 h. Fermentation condition:
Lactobacillus lactis-11, 5-L stirred fermentor, agitation speed of 100 rpm, 42 1 C, feeding glucose solution, recycled calcium carbonate as neutralizing agent).

Signicant differences were found between the SEM images of


recycled and original calcium carbonate (see Figs. 2 and 3, A). The
particles of original calcium carbonate are angular, and their calcite
crystals display characteristic rhombohedral features and measure
approximately 3 m. The recycled calcium carbonate is spherical
in shape, and their crystals display characteristic irregularity and
measure about 0.5 m. The most notable characteristics of recycled calcium carbonate compared with the original particles are
the growth morphologies of the polymorphs and the smaller crystal
size.
A variety of biomacromolecules, such as polypeptides, proteins and polysaccharides, are found in the fermentation broth of
calcium lactate. These biomacromolecules signicantly inuence
the crystallization of calcium carbonate particles. This inuence of proteins on the precipitation of calcium carbonate has

been previously reported. In the presence of proteins, the crystal structure of calcium carbonate may be changed from regular
rhombohedral to needle-like agglomerates, leaf-like agglomerates
or another irregular shape, and the particle morphology may be
changed from angular to rounded or another shape [1013]. In
addition, some studies have suggested that biomacromolecules
(polysaccharides or proteins) can absorb newly formed activated
calcium carbonate and slow down the particles transition to calcite
nanocrystals through crystallization. Additionally, biomacromolecules can inhibit the growth of calcium carbonate particles
[14,15]. Therefore, the presence of biomacromolecules in the fermentation broth was found to be the key factor that made the
particles of recycled calcium carbonate have more impurities, a
smaller particle size, a higher specic surface area and a smaller
crystal size compared with the original calcium carbonate.

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P.-B. Yang et al. / Biochemical Engineering Journal 98 (2015) 3846

Fig. 3. SEM images of original calcium carbonate at different times during the process of fermentation (A, 0 h; B, 15 h; C, 30 h; D, 45 h; E, 54 h; F, 60 h. Fermentation condition:
Lactobacillus lactis-11, 5-L stirred fermentor, agitation speed of 100 rpm, 42 1 C, feeding glucose solution, original calcium carbonate as neutralizing agent).

To summarize, many differences in the composition, particle


size, specic surface area and microstructure between recycled
and original calcium carbonate were measured and observed in
this study. However, these factors alone cannot fully explain why
the use of recycled calcium carbonate instead of original calcium
carbonate as the neutralizing agent yields a more favorable fermentation result. The effects of these two types of calcium carbonate
on lactic acid fermentation should be further investigated.
3.3. Effect of pH
The pH value of the broth is one of the important parameters in
the process of lactic acid fermentation. In general, the optimal pH
of lactic acid bacteria is 6.07.0 [9]. At a low pH value, lactic acid
production would be inhibited due to the increased presence of free
lactic acid. Therefore, the ability of neutralizing agents to adjust the
pH of the broth is the key factor in lactic acid production.
As shown in Fig. 1A and B, recycled calcium carbonate can maintain a higher pH value in the fermentation process (above 5.1

at the end of fermentation) under the condition of high lactate


concentration (143 g L1 ). Conversely, the pH value of the broth
decreased to less than 4.8 under the condition of low lactate concentration (108 g L1 ) when original calcium carbonate was used as
the neutralizing agent; thus, the pH-maintenance ability of recycled calcium carbonate was better than that of original calcium
carbonate.
This phenomenon can be attributed to the difference in the specic surface area between these two types of calcium carbonate.
The specic surface areas of original and recycled calcium carbonate are 0.5615 m2 g1 and 8.190 m2 g1 , respectively. The specic
surface area of recycled calcium carbonate is 14.586-fold greater
than the specic surface area of original calcium carbonate. A recent
study revealed that one of the main factors that affect the reaction
rate between stone particles (natural magnesite and colemanite)
and acid solutions (acetic acid and oxalic acid) is the specic surface
area of the particles [16,17]. The study showed that the dissolution rate (reaction rate between stone particles and acid solution)
increased with increases in the specic surface area. A similar con-

P.-B. Yang et al. / Biochemical Engineering Journal 98 (2015) 3846

clusion can be drawn for the reaction between calcium carbonate


and lactic acid solution. Compared with original calcium carbonate,
recycled calcium carbonate can consume more H+ because of the
particles higher specic surface area. Consequently, as lactic acid is
added to the solution, the concentration of H+ in solution is lower,
and the pH value of the solution is higher than that obtained with
original calcium carbonate.
To verify the inuence of the particles ability to maintain the
pH balance, a lactic acid fermentation experiment was performed.
Original calcium carbonate was used as the neutralizing agent as
the pH value of the broth was adjusted by original calcium carbonate and calcium hydroxide solution (25%, w w1 ) under the same
timetable observed in the recycled calcium carbonate pH experiment. The results are shown in Fig. 1C and Table 1. As the pH
value was adjusted, higher cell dry weight (5.766 g L1 ) as well
as higher lactic acid concentration (124 g L1 ) and productivity
(2.38 g L1 h1 ) were obtained, but these results were still lower
10.1%, 13.3% and 12.8% than those obtained using recycled calcium
carbonate, respectively. This nding indicated that the pH value
was an inuential but not the only factor.
3.4. Effect of nitrogen in recycled calcium carbonate
The nitrogen source is an important factor in lactic acid fermentation by L. lactis. There is little nitrogen in recycled calcium
carbonate. Therefore, the inuence of nitrogen on lactic acid fermentation performance should be researched.
As shown in Section 3.2, the total nitrogen and amino nitrogen contents in recycled calcium carbonate were 0.08% and 0.03%,
respectively. Two-hundred-and-forty grams of calcium carbonate
were added to the broth for each batch. More than 0.192 g of
nitrogen (including 0.072 g of amino nitrogen) was found in the
fermentation system when using recycled calcium carbonate as
the neutralizing agent. This nitrogen was released slowly during
the fermentation due to the reaction between lactic acid and recycled calcium carbonate. Although this portion of nitrogen accounts
for only 2% of the total nitrogen in the broth, it could promote the
growth and activity of L. lactis-11 [18].
To test the effect of this portion of nitrogen on lactic acid fermentation, an experiment in which more nitrogen was added and
used original calcium carbonate was used as the neutralizing agent
was performed. In this experiment, a solution of 1.102 g of yeast
extract and 0.427 g of tryptone (50 mL containing 0.192 g of total
nitrogen and 0.072 g of amino nitrogen) was partly added to the
fermentation system at different times based on the dissolution
rate of recycled calcium carbonate, as shown in Fig. 1A. The times
and volumes of the solution throughout the experiment are shown
in Table 2. The results are shown in Fig. 1D and Table 1. The resulting
cell dry weight, lactic acid concentration and fermentation productivity were only higher 0.9%, 5.56% and 5.85% than those obtained
with original calcium carbonate, respectively.
To further understand the effect of nitrogen, an experiment with
recycled calcium carbonate and a reduced volume of nitrogen was
performed. The results are shown in Fig. 1E and Table 1. Compared
with the results observed with only recycled calcium carbonate,
the fermentation performance was slightly poorer.
These results showed that the nitrogen in recycled calcium carbonate had some promoting effect for lactic acid fermentation but
is not the primary cause.
3.5. Interaction of calcium carbonate particles and microbes
3.5.1. Change of calcium carbonate particles during the process of
fermentation
The ability of recycled calcium carbonate to adjust the pH and
produce nitrogen could promote the production of lactic acid. To

43

Table 2
The adding time and volume of nitrogen solution in the experiment of testing
the Effect of nitrogen in recycled calcium carbonate (Nitrogen solution was 50 mL
containing 0.192 g of total nitrogen and 0.072 g of amino nitrogen. Fermentation
condition: Lactobacillus lactis-11, 5-L stirred fermentor, agitation speed of 100 rpm,
42 1 C, feeding glucose solution, original calcium carbonate as neutralizing agent).
Time (h)

Volume (mL)

Total nitrogen (mg)

Amino nitrogen (mg)

12
15
18
21
24
27
30
33
36
39
42
45
48
51
54
57

7.00
2.10
3.50
1.05
2.45
2.80
2.10
2.10
4.90
2.80
2.80
4.20
4.90
2.80
2.45
2.10

26.85
8.060
13.43
4.030
9.400
10.74
8.060
8.060
18.80
10.74
10.74
16.11
18.80
10.74
9.400
8.060

10.07
3.02
5.03
1.51
3.52
4.03
3.02
3.02
7.05
4.03
4.03
6.04
7.05
4.03
3.52
3.02

verify this theory, another experiment was performed using original calcium carbonate while adjusting the pH and adding additional
nitrogen. The strategies of adjusting the pH and adding nitrogen are
the same as those described in Sections 3.3 and 3.4, respectively.
The results are shown in Fig. 1F and Table 1. The lactic acid concentration and productivity were 130 g L1 and 2.50 g L1 h1 , which
are lower 9.1% and 8.42% than the results observed using recycled
calcium carbonate, respectively. This nding indicated that other
factors are likely work, such as the interaction of calcium carbonate
particles and microbes.
The surface morphology of calcium carbonate particles was
observed during these experiments (see Fig. 2 and Fig. 3). Interestingly, the surface changes of these two types of calcium carbonate
particles during fermentation are completely different. As shown in
Fig. 2, an interesting phenomenon occurred: the recycled calcium
carbonate particles were corroded from one face (Fig. 2B and C), further corroded to obtain a hole (Fig. 2D) and disintegrated into small
debris (Fig. 2E and F). In contrast, the smooth surface of original calcium carbonate became very rough during lactic acid fermentation
(see Fig. 3) but did not form any holes. This is likely the result of
corrosion from the surface of the particles by lactic acid produced
by L. lactis-11.
3.5.2. Evidences of interaction
The interaction between recycled calcium carbonate and L.
lactis-11 was not single and casual. Several similar holes were
observed in different lactic acid fermentation experiments using
recycled calcium carbonate as neutralizing agents (see Fig. 4). Furthermore, direct proof that L. lactis-11 can enter the holes of the
recycled calcium carbonate particles was obtained. As shown in
Fig. 4C, there is a round object in the particle, and the surface looks
different than the surface of the calcium carbonate particles. Therefore, the surface element of the round object was analyzed using
an X-ray energy dispersive spectrometer. The results are shown in
Fig. 5. The carbon-to-oxygen ratio is 59.58:35.92, which is very close
to the carbon-to-oxygen ratio (C:O = 60:36.6) of peptidoglycan, the
main component of the outer cell wall of lactic acid bacteria.
Another factor serves as evidence for the interaction between
recycled calcium carbonate particles and microbes. This evidence
is the difference between two measurements of the cell dry weight.
One result of the cell dry weight (CDW1 ) was obtained from
measurement of the suspension (including the remaining calcium
carbonate particles after dissolution by diluted hydrochloric acid),
and the other result (CDW2 ) was obtained from measurement of

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P.-B. Yang et al. / Biochemical Engineering Journal 98 (2015) 3846

Fig. 4. SEM images of the holes in recycled calcium carbonate particles (Fermentation condition: Lactobacillus lactis-11, 5-L stirred fermentor, agitation speed of 100 rpm,
42 1 C, feeding glucose solution, original calcium carbonate as neutralizing agent).

the supernatant (not including the remaining calcium carbonate


after dissolution by a small amount of diluted hydrochloric acid).
The results are shown in Fig. 1. When using recycled calcium carbonate as the neutralizing agent, CDW1 was higher than CDW2 (see
Fig. 1A and E). When original calcium carbonate was used, CDW1
was very close to CDW2 (see Fig. 1BD and F). This nding indicated that part of the L. lactis-11 cell had entered the particles of
recycled calcium carbonate or had been absorbed onto the surface,
a phenomenon that was not observed in experiments using original
calcium carbonate.
How were these holes in the particles of recycled calcium carbonate formed? The preliminary conclusion is that these holes were
formed by L. lactis-11. A similar phenomenon can be observed in
bioleaching research, which shows that the adhesion of a microbe
and a particle is the primary prerequisite of interaction. In general,
the inuential factors of adhesion include the surface properties
and the effect of solution chemistry and physics [1921]. During
bioleaching, a microorganism (Acidithiobacillus ferrooxidans) can
attach to the mineral particle surface (chalcopyrite), and a number of holes that were similar to the shape of Acidithiobacillus
ferrooxidans were formed after 48 days of bio-oxidation [22,23].

Analogously, L. lactis-11 can be absorbed into the surface of recycled calcium carbonate. Some research studies have indicated that
bacteria like to be absorbed onto the rough surface of a particle or on
the surface of a particle with a high surface area because of reduced
surface energy [2426]. The particles of recycled calcium carbonate
have these advantages (see Fig. 2A and Section 3.2). Unlike chalcopyrite, calcium carbonate can be easily corroded by lactic acid.
Additionally, L. lactis-11 can produce lactic acid directly. Therefore,
L. lactis-11 can make these holes within the particles of recycled
calcium carbonate in a relatively short period of time.

3.5.3. Benecial effect of interaction to producing lactic acid


It has been reported that mineral particles can benet the
growth and activity of microorganisms. Particles are a ne place
for microbial growth and reproduction. Mineral particles tend to
absorb some organic matter, which could then be used by microbes
to synthesize its own composition and transform into energy [27].
Microbes can also assimilate magnesium and calcium from mineral
grains, which could activate many enzymatic reactions or maintain
the normal physiological state of microorganisms [28]. In addition,

P.-B. Yang et al. / Biochemical Engineering Journal 98 (2015) 3846

45

glucose in the hole was consumed by the cell, resulting in a reduction in the glucose concentration in this small space. This small
space with a low glucose concentration could exist for a relatively
long period of time because the material exchange is limited. A
concentration gradient of glucose between the inside and outside
of the hole was formed. Therefore, the substrate inhibition effect
to L. lactis-11 was reduced. The material exchange between the
inside and outside of the hole was not cut off completely; thus, the
glucose outside the hole could diffuse to the inside under the impetus of the concentration difference.
4. Conclusions
Recycled calcium carbonate has a signicant effect on lactic acid
fermentation. The volume of lactic acid produced and the cell dry
weight increased by 33.17% and 19.24%, respectively, using recycled calcium carbonate as the neutralizing agent. This increase
could be attributed to the characteristics of recycled calcium carbonate, such as its anomalous and small crystalline grains (200 nm),
small particle size (4.682 m), high surface area (8.190 m2 g1 )
and presence of some nitrogen. These characteristics give recycled calcium carbonate the ability to maintain a higher pH, thus
promoting the growth of L. lactis-11 and accelerating lactic acid
production. In addition, the direct interaction between L. lactis-11
and recycled calcium carbonate was conrmed by macroscopic and
microscopic evidence, and this direct interaction may provide a
favorable microenvironment to the cell.
Acknowledgment
This work was supported by the program of Science and Technology Service Network Initiative of the Chinese Academy of
Sciences (KFJ-EW-STS077-RW3).
References

Fig. 5. SEM images and results of the surface elements analysis of Lactobacillus lactis11 cell in the hole of a recycled calcium carbonate particle (Fermentation condition:
Lactobacillus lactis-11, 5-L stirred fermentor, agitation speed of 100 rpm, 42 1 C,
feeding glucose solution, original calcium carbonate as neutralizing agent).

microorganisms can maintain a microenvironment relatively well


[29].
Similarly, these holes within the particles of recycled calcium
carbonate can provide a comparatively favorable microenvironment for the L. lactis-11 cell. First, the holes of calcium carbonate
particle like the big and hard shell of the L. lactis-11 cell, which
is located in the hole. This type of big and hard shell could make
the cell maintain a high activity level, which could protect the cell
from the effects of grinding and shearing forces. Second, once lactic acid produced by L. lactis-11 is released into the extracellular
solution, this part of lactic acid could be rapidly consumed by the
calcium carbonate around it and could thus cause a reduction in
the concentration of free lactic acid in the solution around the
cell. As a result, the feedback inhibition effect of free lactic acid
could be reduced, and the pH value of the cell microenvironment
could increase. Finally, the L. lactis-11 cells in the holes were in a
semi-closed environment, which limits the material exchange. The

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