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 acidfermentation performance of Lactobacillus lactis

Peng-Bo Yanga,b, Yuan Tiana, Qian Wanga, Wei Conga,

a National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, PR Chinab 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 2014Received in revised form 4 January 2015Accepted 18 February 2015Available online 20 February 2015

Keywords:Lactic acidFermentationRecycled calcium carbonateDirect interactionAfnityAdsorption

a b s t r a c t

The effect of two different types of calcium carbonate on lactic acid fermentation was investigated in thisstudy. The results showed that the fermentation performance of calcium carbonate greatly improvedwhen the recycled particle was used instead of the original particle. The improved fermentation wasevidenced by the pH of the broth, the enhanced production of lactic acid (increase of 33.17%) and theenhanced cell dry weight (CDW, increase of 19.24%). Analysis of the composition and structure of cal-cium carbonate revealed that the recycled compound possesses several unique features that allow it tomaintain a higher pH and provide nitrogen during fermentation. The experimental results indicated thata higher pH value and the addition of nitrogen could increase the fermentation performance, but thesefactors were not sufcient to explain the original ndings. Macroscopic and microscopic studies con-rmed that direct interactions, such as absorption and particle entrance, occurred between Lactobacilluslactis-11 and the recycled calcium carbonate particles. These direct interactions may provide a favorablemicroenvironment 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 tex-tile manufacturing [1]. Fermentation has recently been consideredthe main method for producing lactic acid [2,3]. During lactic acidfermentation, the pH value of the broth decreases as the concentra-tion of undissociated lactic acid increases. This condition inhibitsthe ability of microbes to produce lactic acid due to the feedbackinhibition effect. Consequently, to obtain a higher production rateof lactic acid, a neutralizer should be added to the fermentationbroth to stabilize its pH value [4].

According to previous studies, calcium carbonate, calciumhydroxide, ammonia, sodium hydroxide or potassium hydroxidecould be used as neutralizing agents in the lactic acid fermen-tation process [5,6]. Ammonia, sodium hydroxide and potassiumhydroxide are rapid and effective neutralizers and can thereforestabilize the pH value of the broth without producing calciumsulfate as a byproduct. However, the nal acid and cell con-centrations 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).

lactic acid toward microbial cells. Calcium alkali is a temperateneutralizer for lactic acid bacteria and yields a high lactic acidconcentration when added to the broth [7]. However, solid waste(calcium sulfate) is unavoidably produced in the extraction pro-cess.

To solve this problem, a new technology for the productionof lactic acid was developed using calcium carbonate as a neu-tralizer without yielding calcium sulfate as a byproduct [8]. Thecore step of this technology was a displacement reaction that canconvert calcium lactate into soluble lactate and calcium carbon-ate (recycled calcium carbonate). To ensure that the fermentationprocess was environmentally friendly and economical, the recy-cled calcium carbonate can be re-used as the neutralizer in thenext fermentation batch. An interesting nding from the experi-ments was that lactic acid bacteria appeared to be more interestedin the recycled calcium carbonate compared with the original cal-cium carbonate. However, the mechanism for this phenomenon isunknown.

This study provides the rst demonstration of the effects oftwo types of calcium carbonate on lactic acid fermentation. Theconstitution, surface morphology and particle size were com-pared between the calcium carbonate types, and the relationshipbetween the molecular characteristics and experimental results isdiscussed.

http://dx.doi.org/10.1016/j.bej.2015.02.0231369-703X/ 2015 Elsevier B.V. All rights reserved.

P.-B. Yang et al. / Biochemical Engineering Journal 98 (2015) 3846 39

Table 1Lactic acid fermentation with different conditions (CaCO3, nitrogen source and pH) by Lactobacillus lactis-11 (Fermentation condition: 5-L stirred fermentor, agitation speedof 100 rpm, 421 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 after12h(g L1 h1)

Recycled CaCO3 60 6.414 143 0.978 2.73Original CaCO3 60 5.379 108 0.987 2.05Original CaCO3 and pH adjustment 60 5.766 124 0.982 2.38Original CaCO3 and adding N 60 5.426 114 0.985 2.17Recycled CaCO3 and reducing N 60 6.306 140 0.979 2.69Original CaCO3, pH adjustment and adding N 60 5.878 130 0.980 2.50

2. Methods

2.1. Microorganism and media

Lactobacillus lactis-11 (providedbyShandongUniversity, China),a lactic acid-producing strain of the bacteria, was grown on de ManRogosa Sharpe (MRS) [9] medium. The seed culture was grownin MRS broth at 42 C with shaking at 100 rpm for 12h, and theinoculation size was 10% (v v1) in all of the experiments. The fer-mentation broth consisted of (per liter of distilled water) 5 g ofyeast, 10 gofpeptone, 10gofbeef extract, 10 gofNaCl, 5 gof sodiumacetate, 2 g of triammoniumcitrate, 0.4 g ofMgSO4, 0.01 g ofMnSO4and 20g of glucose.

2.2. Batch fermentation

The batch cultures were performed in a 5-L stirred fermentor(BaoXing, Shanghai, China). The volumeof initialmediumwas3.0 L.No air was introduced to the cultures, and an agitation speed of100 rpmwasemployed to ensure thehomogeneity of the fermenta-tion broth. The culture temperaturewas set to 421 C. The carbonsource (800g L1 glucose solution) was fed into the fermentor dur-ing the fermentation, and the feeding speed was set according tothe concentration of residual sugar in broth. The neutralizing agentwas calcium carbonate, which was added to the broth before fer-mentation. The calcium carbonate addition in one batch was 240g,and the ratio of glucose to calcium carbonate was about 3:2 (w/w).

2.3. Preparation of recycled calcium carbonate

The recycled calciumcarbonatewas obtained from the displace-ment reactionbetweencalcium lactate in thebroth andammoniumcarbonate. First, the freshly fermented broth was centrifuged at4000 g for 25min, and the supernatant was then poured intoan airtight agitation tank. Afterward, ammonium carbonate wassupplied to the supernatant at a ratio of 1:1 (molmol1), and thesupernatantwas agitated at 200 rpmand room temperature for 3h.Finally, an additional centrifugation was conducted at 4000 g for5min, and the residue obtained was used as the neutralizing agentin this study.

2.4. Analysis

2.4.1. Determination of the microbe and substanceconcentrations

Thebiomass concentrationwas expressed as the cell dryweight,whichwasdeterminedbymeasuring the optical density (OD) of thebroth at 620nm. The calcium carbonate particle should react afterthe addition of diluted hydrochloric acid. The optical density wasproportional to the cell dry weight, and one OD unit correspondsto 0.516g L1 of biomass.

The glucose concentration was determined using a SBA-40Cbiosensor analyzer (Institute of biology, Shangdong ProvinceAcademy of Sciences, P.R. China).

Lactic acid was measured by high-performance liquid chro-matography (HPLC) [5].

The nitrogen content was measured using a Flash EA 1112 ele-mental analyzer (Thermo Fisher Scientic Inc., USA). The sample ofcalcium carbonate was dissolved in dilute hydrochloric acid. Eachsample measurement was performed in duplicate, and the averagevalue is reported. The difference between the values was alwaysless than 5%.

The content of amino nitrogen was measured by the Coomassiebrilliant blue method.

2.4.2. Electron microscopy and surface elemental analysisImages of the calcium carbonate particles were taken at

10kV with a scanning electron microscope (SEM; S-4800, HitachiHigh-Technologies Corporation, Japan). Elemental analyses wereperformed using an X-ray energy dispersive spectrometer (EDS)(EDAX9100, JEOL Ltd., Japan).

2.4.3. Particle size determinationTheparticle size distributionwas analyzedusing aMalvinHydro

2000Mu laser particle size analyzer. The sampleswere dispersed inwater by ultrasonic treatment for 2min before the measurements.

2.4.4. Specic surface area determinationThe specic surface area was determined using N2 with a spe-

cic surface area analyzer (Autosorb-1, Quantachrome, USA).

3. Results and discussion

3.1. Different fermentation performances obtained using recycledand original calcium carbonate

Batch fermentationswereperformedunder the sameconditionswith the exception of the neutralizing agents. In the different batchfermentations, recycled and original calcium carbonate were usedas neutralizing agents. As shown in Fig. 1A andB, the use of recycledcalcium carbonate as the neutralizing agent increased the concen-tration of lactic acid from 108g L1 to 143g L1 over the same timeperiod and under the same fermentation conditions as the exper-iment with original calcium carbonate. The presence of recycledcalcium carbonate was favorable for the growth of L. lactis-11, asevidenced by an increase in the cell dry weight from 5.379g L1 to6.414g L1 (see Table 1). The ability of recycled calcium carbonateto adjust the pH of the solution was better than that of originalcalcium carbonate.

The data from the lactic acid accumulation stage (after 12h)were selected for further analysis. As shown in Table 1, the produc-tion rate of lactic acid obtained using recycled calcium carbonateas the neutralizing agent reached 2.73g L1 h1, which was 33.17%

40 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 andadjusting pH by the addition of calcium hydroxide solution; D, original calcium carbonate and adding more nitrogen; E, recycled calcium carbonate and reducing part ofnitrogen; F, original calcium carbonate, adjusting pH by the addition of calcium hydroxide solution and adding more nitrogen. , lactic acid; , glucose; , pH; , cell dryweight of suspension (CDW1); , cell dry weight of supernatant (CDW2)].

higher than the production rate of lactic acid obtainedwith originalcalcium carbonate (2.05g L1 h1).

This result is due to the type of calcium carbonate used. Con-sequently, the difference between these two types of calciumcarbonate should be further investigated.

3.2. Structural features of recycled and original calciumcarbonate

The composition, particle size and specic surface area of recy-cled and original calcium carbonate are compared. The results

showed that recycled calcium carbonate produced nitrogen duringfermentation and had a smaller particle size and a higher spe-cic surface area compared with the original calcium carbonate.The total nitrogen and amino nitrogen contents in recycled cal-cium carbonate were found to be 0.08% and 0.03%, respectively.In contrast, the nitrogen content in the original calcium carbon-ate was below the detection limit (less than 0.01%). The particlesizes of recycled and original calcium carbonate were 4.682mand 12.431m, respectively, and the specic surface areas of recy-cled and original calcium carbonate were found to be 8.190m2 g1

and 0.5615m2 g1, respectively.

P.-B. Yang et al. / Biochemical Engineering Journal 98 (2015) 3846 41

Fig. 2. SEM images of recycled calcium carbonate at different times during the process of fermentation (A, 0h; B, 12h; C, 27h; D, 39h; E, 51h; F, 60h. Fermentation condition:Lactobacillus lactis-11, 5-L stirred fermentor, agitation speed of 100 rpm, 421 C, feeding glucose solution, recycled calcium carbonate as neutralizing agent).

Signicant differences were found between the SEM images ofrecycled and original calcium carbonate (see Figs. 2 and 3, A). Theparticles of original calcium carbonate are angular, and their calcitecrystals display characteristic rhombohedral features and measureapproximately 3m. The recycled calcium carbonate is sphericalin shape, and their crystals display characteristic irregularity andmeasure about 0.5m. The most notable characteristics of recy-cled calcium carbonate compared with the original particles arethe growthmorphologies of thepolymorphs and the smaller crystalsize.

A variety of biomacromolecules, such as polypeptides, pro-teins and polysaccharides, are found in the fermentation broth ofcalcium lactate. These biomacromolecules signicantly inuencethe crystallization of calcium carbonate particles. This inu-ence of proteins on the precipitation of calcium carbonate has

been previously reported. In the presence of proteins, the crys-tal structure of calcium carbonate may be changed from regularrhombohedral to needle-like agglomerates, leaf-like agglomeratesor another irregular shape, and the particle morphology may bechanged from angular to rounded or another shape [1013]. Inaddition, some studies have suggested that biomacromolecules(polysaccharides or proteins) can absorb newly formed activatedcalciumcarbonate and slowdown the particles transition to calcitenanocrystals through crystallization. Additionally, biomacro-molecules can inhibit the growth of calcium carbonate particles[14,15]. Therefore, the presence of biomacromolecules in the fer-mentation broth was found to be the key factor that made theparticles of recycled calcium carbonate have more impurities, asmaller particle size, a higher specic surface area and a smallercrystal size compared with the original calcium carbonate.

42 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, 0h; B, 15h; C, 30h; D, 45h; E, 54h; F, 60h. Fermentation condition:Lactobacillus lactis-11, 5-L stirred fermentor, agitation speed of 100 rpm, 421 C, feeding glucose solution, original calcium carbonate as neutralizing agent).

To summarize, many differences in the composition, particlesize, specic surface area and microstructure between recycledand original calcium carbonate were measured and observed inthis study. However, these factors alone cannot fully explain whythe use of recycled calcium carbonate instead of original calciumcarbonate as theneutralizing agent yields amore favorable fermen-tation result. The effects of these two types of calcium carbonateon lactic acid fermentation should be further investigated.

3.3. Effect of pH

The pH value of the broth is one of the important parameters inthe process of lactic acid fermentation. In general, the optimal pHof lactic acid bacteria is 6.07.0 [9]. At a low pH value, lactic acidproductionwouldbe inhibiteddue to the increasedpresence of freelactic acid. Therefore, the ability of neutralizing agents to adjust thepH of the broth is the key factor in lactic acid production.

As shown in Fig. 1A and B, recycled calcium carbonate canmain-tain a higher pH value in the fermentation process (above 5.1

at the end of fermentation) under the condition of high lactateconcentration (143g L1). Conversely, the pH value of the brothdecreased to less than 4.8 under the condition of low lactate con-centration (108g L1)when original calcium carbonatewas used asthe neutralizing agent; thus, the pH-maintenance ability of recy-cled calcium carbonate was better than that of original calciumcarbonate.

This phenomenon can be attributed to the difference in the spe-cic surface area between these two types of calcium carbonate.The specic surface areas of original and recycled calcium carbon-ate are 0.5615m2 g1 and 8.190m2 g1, respectively. The specicsurface area of recycled calcium carbonate is 14.586-fold greaterthan the specic surface areaof original calciumcarbonate. A recentstudy revealed that one of the main factors that affect the reactionrate between stone particles (natural magnesite and colemanite)and acid solutions (acetic acid andoxalic acid) is the specic surfacearea of the particles [16,17]. The study showed that the dissolu-tion 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 43

clusion can be drawn for the reaction between calcium carbonateand lactic acid solution. Comparedwith original calcium carbonate,recycled calcium carbonate can consume more H+ because of theparticles higher specic surface area. Consequently, as lactic acid isadded to the solution, the concentration of H+ in solution is lower,and the pH value of the solution is higher than that obtained withoriginal calcium carbonate.

To verify the inuence of the particles ability to maintain thepH balance, a lactic acid fermentation experiment was performed.Original calcium carbonate was used as the neutralizing agent asthe pH value of the broth was adjusted by original calcium carbon-ate and calcium hydroxide solution (25%, ww1) under the sametimetable observed in the recycled calcium carbonate pH exper-iment. The results are shown in Fig. 1C and Table 1. As the pHvalue was adjusted, higher cell dry weight (5.766g L1) as wellas higher lactic acid concentration (124g L1) and productivity(2.38g L1 h1) were obtained, but these results were still lower10.1%, 13.3% and 12.8% than those obtained using recycled calciumcarbonate, respectively. This nding indicated that the pH valuewas 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 fer-mentation by L. lactis. There is little nitrogen in recycled calciumcarbonate. Therefore, the inuence of nitrogen on lactic acid fer-mentation performance should be researched.

As shown in Section 3.2, the total nitrogen and amino nitro-gen contents in recycled calcium carbonate were 0.08% and 0.03%,respectively. Two-hundred-and-forty grams of calcium carbonatewere added to the broth for each batch. More than 0.192g ofnitrogen (including 0.072g of amino nitrogen) was found in thefermentation system when using recycled calcium carbonate asthe neutralizing agent. This nitrogen was released slowly duringthe fermentation due to the reaction between lactic acid and recy-cled calcium carbonate. Although this portion of nitrogen accountsfor only 2% of the total nitrogen in the broth, it could promote thegrowth and activity of L. lactis-11 [18].

To test the effect of this portion of nitrogen on lactic acid fer-mentation, an experiment in which more nitrogen was added andused original calcium carbonate was used as the neutralizing agentwas performed. In this experiment, a solution of 1.102g of yeastextract and 0.427g of tryptone (50mL containing 0.192g of totalnitrogen and 0.072g of amino nitrogen) was partly added to thefermentation system at different times based on the dissolutionrate of recycled calcium carbonate, as shown in Fig. 1A. The timesand volumes of the solution throughout the experiment are showninTable 2. The results are shown in Fig. 1DandTable 1. The resultingcell dry weight, lactic acid concentration and fermentation produc-tivity were only higher 0.9%, 5.56% and 5.85% than those obtainedwith original calcium carbonate, respectively.

To further understand the effect of nitrogen, an experimentwithrecycled calcium carbonate and a reduced volume of nitrogen wasperformed. The results are shown in Fig. 1E and Table 1. Comparedwith the results observed with only recycled calcium carbonate,the fermentation performance was slightly poorer.

These results showed that the nitrogen in recycled calcium car-bonate had some promoting effect for lactic acid fermentation butis not the primary cause.

3.5. Interaction of calcium carbonate particles and microbes

3.5.1. Change of calcium carbonate particles during the process offermentation

The ability of recycled calcium carbonate to adjust the pH andproduce nitrogen could promote the production of lactic acid. To

Table 2The adding time and volume of nitrogen solution in the experiment of testingthe Effect of nitrogen in recycled calcium carbonate (Nitrogen solution was 50mLcontaining 0.192g of total nitrogen and 0.072g of amino nitrogen. Fermentationcondition: Lactobacillus lactis-11, 5-L stirred fermentor, agitation speed of 100 rpm,421 C, feedingglucose solution, original calciumcarbonate asneutralizing agent).

Time (h) Volume (mL) Total nitrogen (mg) Amino nitrogen (mg)

12 7.00 26.85 10.0715 2.10 8.060 3.0218 3.50 13.43 5.0321 1.05 4.030 1.5124 2.45 9.400 3.5227 2.80 10.74 4.0330 2.10 8.060 3.0233 2.10 8.060 3.0236 4.90 18.80 7.0539 2.80 10.74 4.0342 2.80 10.74 4.0345 4.20 16.11 6.0448 4.90 18.80 7.0551 2.80 10.74 4.0354 2.45 9.400 3.5257 2.10 8.060 3.02

verify this theory, another experiment was performed using origi-nal calciumcarbonatewhile adjusting thepHandadding additionalnitrogen. The strategies of adjusting the pHand adding nitrogen arethe 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 concen-tration and productivity were 130g L1 and 2.50g L1 h1, whichare lower 9.1% and 8.42% than the results observed using recycledcalcium carbonate, respectively. This nding indicated that otherfactors are likelywork, such as the interaction of calcium carbonateparticles and microbes.

The surface morphology of calcium carbonate particles wasobserved during these experiments (see Fig. 2 and Fig. 3). Interest-ingly, the surface changes of these two types of calcium carbonateparticles during fermentation are completely different. As shown inFig. 2, an interesting phenomenon occurred: the recycled calciumcarbonateparticleswere corroded fromone face (Fig. 2B andC), fur-ther corroded to obtain a hole (Fig. 2D) and disintegrated into smalldebris (Fig. 2E and F). In contrast, the smooth surface of original cal-cium carbonate became very rough during lactic acid fermentation(see Fig. 3) but did not form any holes. This is likely the result ofcorrosion from the surface of the particles by lactic acid producedby L. lactis-11.

3.5.2. Evidences of interactionThe interaction between recycled calcium carbonate and L.

lactis-11 was not single and casual. Several similar holes wereobserved in different lactic acid fermentation experiments usingrecycled calcium carbonate as neutralizing agents (see Fig. 4). Fur-thermore, direct proof that L. lactis-11 can enter the holes of therecycled calcium carbonate particles was obtained. As shown inFig. 4C, there is a round object in the particle, and the surface looksdifferent than the surface of the calcium carbonate particles. There-fore, the surface element of the round object was analyzed usingan X-ray energy dispersive spectrometer. The results are shown inFig. 5. Thecarbon-to-oxygenratio is59.58:35.92,which isverycloseto the carbon-to-oxygen ratio (C:O=60:36.6) of peptidoglycan, themain component of the outer cell wall of lactic acid bacteria.

Another factor serves as evidence for the interaction betweenrecycled calcium carbonate particles and microbes. This evidenceis the difference between twomeasurements of the cell dryweight.One result of the cell dry weight (CDW1) was obtained frommeasurement of the suspension (including the remaining calciumcarbonate particles after dissolution by diluted hydrochloric acid),and the other result (CDW2) was obtained from measurement of

44 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,421 C, feeding glucose solution, original calcium carbonate as neutralizing agent).

the supernatant (not including the remaining calcium carbonateafter dissolution by a small amount of diluted hydrochloric acid).The results are shown in Fig. 1. When using recycled calcium car-bonate as the neutralizing agent, CDW1 was higher than CDW2 (seeFig. 1A and E). When original calcium carbonate was used, CDW1was very close to CDW2 (see Fig. 1BD and F). This nding indi-cated that part of the L. lactis-11 cell had entered the particles ofrecycled calcium carbonate or had been absorbed onto the surface,a phenomenon thatwas not observed in experiments using originalcalcium carbonate.

How were these holes in the particles of recycled calcium car-bonate formed?Thepreliminary conclusion is that theseholeswereformed by L. lactis-11. A similar phenomenon can be observed inbioleaching research, which shows that the adhesion of a microbeand a particle is the primary prerequisite of interaction. In general,the inuential factors of adhesion include the surface propertiesand the effect of solution chemistry and physics [1921]. Duringbioleaching, a microorganism (Acidithiobacillus ferrooxidans) canattach to the mineral particle surface (chalcopyrite), and a num-ber of holes that were similar to the shape of Acidithiobacillusferrooxidans were formed after 48 days of bio-oxidation [22,23].

Analogously, L. lactis-11 can be absorbed into the surface of recy-cled calcium carbonate. Some research studies have indicated thatbacteria like tobeabsorbedonto the roughsurfaceof aparticle oronthe surface of a particle with a high surface area because of reducedsurface energy [2426]. The particles of recycled calcium carbonatehave these advantages (see Fig. 2A and Section 3.2). Unlike chal-copyrite, 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 recycledcalcium carbonate in a relatively short period of time.

3.5.3. Benecial effect of interaction to producing lactic acidIt has been reported that mineral particles can benet the

growth and activity of microorganisms. Particles are a ne placefor microbial growth and reproduction. Mineral particles tend toabsorb some organicmatter, which could then be used bymicrobesto synthesize its own composition and transform into energy [27].Microbes can also assimilatemagnesium and calcium frommineralgrains, which could activate many enzymatic reactions or maintainthe normal physiological state of microorganisms [28]. In addition,

P.-B. Yang et al. / Biochemical Engineering Journal 98 (2015) 3846 45

Fig. 5. SEMimages and results of the surface elements analysis of Lactobacillus lactis-11 cell in the hole of a recycled calcium carbonate particle (Fermentation condition:Lactobacillus lactis-11, 5-L stirred fermentor, agitation speed of 100 rpm, 421 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 calciumcarbonate can provide a comparatively favorable microenviron-ment for the L. lactis-11 cell. First, the holes of calcium carbonateparticle like the big and hard shell of the L. lactis-11 cell, whichis located in the hole. This type of big and hard shell could makethe cell maintain a high activity level, which could protect the cellfrom the effects of grinding and shearing forces. Second, once lac-tic acid produced by L. lactis-11 is released into the extracellularsolution, this part of lactic acid could be rapidly consumed by thecalcium carbonate around it and could thus cause a reduction inthe concentration of free lactic acid in the solution around thecell. As a result, the feedback inhibition effect of free lactic acidcould be reduced, and the pH value of the cell microenvironmentcould increase. Finally, the L. lactis-11 cells in the holes were in asemi-closed environment, which limits the material exchange. The

glucose in the hole was consumed by the cell, resulting in a reduc-tion in the glucose concentration in this small space. This smallspace with a low glucose concentration could exist for a relativelylong period of time because the material exchange is limited. Aconcentration gradient of glucose between the inside and outsideof the hole was formed. Therefore, the substrate inhibition effectto L. lactis-11 was reduced. The material exchange between theinside and outside of the hole was not cut off completely; thus, theglucose outside the hole could diffuse to the inside under the impe-tus of the concentration difference.

4. Conclusions

Recycled calcium carbonate has a signicant effect on lactic acidfermentation. The volume of lactic acid produced and the cell dryweight increased by 33.17% and 19.24%, respectively, using recy-cled calcium carbonate as the neutralizing agent. This increasecould be attributed to the characteristics of recycled calcium car-bonate, such as its anomalous and small crystalline grains (200nm),small particle size (4.682m), high surface area (8.190m2 g1)and presence of some nitrogen. These characteristics give recy-cled calcium carbonate the ability to maintain a higher pH, thuspromoting the growth of L. lactis-11 and accelerating lactic acidproduction. In addition, the direct interaction between L. lactis-11and recycled calciumcarbonatewas conrmedbymacroscopic andmicroscopic evidence, and this direct interaction may provide afavorable microenvironment to the cell.

Acknowledgment

This work was supported by the program of Science and Tech-nology Service Network Initiative of the Chinese Academy ofSciences (KFJ-EW-STS077-RW3).

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Effect of different types of calcium carbonate on the lactic acid fermentation performance of Lactobacillus lactis1 Introduction2 Methods2.1 Microorganism and media2.2 Batch fermentation2.3 Preparation of recycled calcium carbonate2.4 Analysis2.4.1 Determination of the microbe and substance concentrations2.4.2 Electron microscopy and surface elemental analysis2.4.3 Particle size determination2.4.4 Specific surface area determination

3 Results and discussion3.1 Different fermentation performances obtained using recycled and original calcium carbonate3.2 Structural features of recycled and original calcium carbonate3.3 Effect of pH3.4 Effect of nitrogen in recycled calcium carbonate3.5 Interaction of calcium carbonate particles and microbes3.5.1 Change of calcium carbonate particles during the process of fermentation3.5.2 Evidences of interaction3.5.3 Beneficial effect of interaction to producing lactic acid

4 ConclusionsAcknowledgmentReferences