Beruflich Dokumente
Kultur Dokumente
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
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)
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.
2.4. 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.
40
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
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).
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.
42
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).
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)
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
44
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).
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.
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).
[1] Z.Y. Zhang, B. Jin, J.M. Kelly, Production of lactic acid from renewable
materials by Rhizopus fungi, Biochem. Eng. J. 35 (2007) 251263.
[2] M.I. Gonzlez, S. lvarez, F. Riera, R. lvarez, Economic evaluation of an
integrated process for lactic acid production from ultraltered whey, J. Food
Eng. 80 (2007) 553561.
[3] Y.J. Wee, H.W. Ryu, Lactic acid production by Lactobacillus sp. RKY2 in a
cell-recycle continuousfermentation using lignocellulosic hydrolyzates as
inexpensive raw materials, Bioresour. Technol. 100 (2009) 42624270.
[4] Y. Zhou, J.M. Domnguez, N. Cao, J. Du, G.T. Tsao, Optimization of l-lactic acid
production from glucose by Rhizopus oryzae ATCC 52,311, Appl. Biochem.
Biotechnol. 77 (1999) 401407.
[5] D.M. Bai, M.Z. Jia, X.M. Zhao, R. Ban, F. Shen, X.G. Li, S.M. Xu, l(+)-lactic acid
production by pellet-from Rhizopus oryzae R1021 in a stirred tank fermentor
l(+)-lactic acid production by pellet-from Rhizopus oryzae R1021 in a stirred
tank fermentor, Chem. Eng. Sci. 58 (2003) 785791.
[6] M.C. Yu, R.C. Wang, C.Y. Wang, K.J. Duan, D.C. Sheu, Enhanced production of
l(+)-lactic acid by oc-form culture of Rhizopus oryzae, J. Chin. Inst. Chem.
Eng. 38 (2007) 223228.
[7] C.Y. Wang, C.T. Lin, D.C. Sheu, C.Y. Liu, l-Lactic acid fermentation by culture of
Rhizopus oryzae using ammonia as neutralizing agent l-Lactic acid
fermentation by culture of Rhizopus oryzae using ammonia as neutralizing
agent, J. Taiwan Inst. Chem Eng. 45 (2014) 663669.
[8] W. Cong, P.B. Yang, L.J. Wang, Y. Zhang, Q. Wang, X. Wu, A method of clean
production of lactic acid. Chinese patent, (2010) Application number:
201010150058.6.
[9] Y. Zhang, W. Cong, S.Y. Shi, Application of a pH feedback-controlled substrate
feeding method in lactic acid production, Appl. Biochem. Biotechnol. 162
(2010) 21492156.
[10] X.H. Guo, A.W. Xu, S.H. Yu, Crystallization of calcium carbonate mineral with
hierarchical structures in DMF solution under control of poly (ethylene
glycol)-b-poly(l-glutamic acid): effects of crystallization temperature and
polymer concentration, Cryst. Growth Des. 8 (2008) 12331242.
[11] Z.P. Zhang, D.M. Gao, H. Zhao, C.G. Xie, G.J. Guan, D.P. Wang, S.H. Yu,
Biomimetic assembly of polypeptide-stabilized CaCO3 nanoparticles, J. Phys.
Chem. B 110 (2006) 86138618.
[12] A. Hernndez-Hernndez, A.B. Rodrguez-Navarro, J. Gmez-Morales, C.
Jimnez-Lopez, Y. Nys, J.M. Garca-Ruiz, Inuence of model globular proteins
with different isoelectric points on the precipitation of calcium carbonate,
Cryst. Growth Des. 8 (2008) 14951502.
46