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Bioresource Technology 99 (2008) 4368–4379

Green Biorefinery: Separation of lactic acid from grass silage juiceby chromatography using neutral polymeric resin

Vu Hong Thang a,b, Senad Novalin a,*

a Department of Food Science and Technology, Vienna University of Natural Resources and Applied Life Sciences (BOKU),

Muthgasse 18, A-1190 Vienna, Austriab Department of Fermentation Technology, Hanoi University of Technology, 1 Dai Co Viet Road, Hanoi, Vietnam

Received 22 June 2006; received in revised form 14 June 2007; accepted 23 August 2007Available online 1 November 2007

Abstract

The aim of this work was to recover lactic acid in undissociated form from grass silage juice. For this aim, chromatographic sepa-ration using neutral polymeric resin Amberlite XAD1600 was investigated. Up to now, there is no hint in the literatures about usingneutral polymeric resin for lactic acid separation from a mixture. Important factors (flow-rate, concentration of feed and loaded volume)that affect separation performance were firstly investigated with model solutions. The obtained results showed that lactic acid solutionswith the purity varying from 93.2% to 99.9% could be obtained at the recovery yields over 99.4%. After that, trials with silage juice werecarried out. Due to the complex composition of the feed, the purity of products decreased to 94% at a recovery yield of 97%. Although99% of inorganic salts and sugars were separated from lactic acid organic acids in general and acetic acid in particular caused a purityproblem. It seems that organic acids could not be separated from lactic acid by neutral resin Amberlite XAD1600. Besides the organicacid problem, some amino acids were remained in the products as impurities.� 2007 Elsevier Ltd. All rights reserved.

Keywords: Grass silage; Lactic acid; Amberlite XAD1600; Preparative chromatography

1. Introduction

Agriculture in many European countries is currentlyundergoing structural changes, which are characterizedby a decrease of livestock and dairy farming. One of theconsequences is an increase of unused grassland biomass(grass) and uncultivated grassland (fallow land), respec-tively. It was estimated that, in the medium term, 750,000tons of dry matter per year would be available from grass-land in Austria (Forschungsforum – BMVIT, 2004). Inno-vative technology concepts for the utilization of thisunused grass can provide the raw material for the manufac-ture of future-oriented products, and thus strengthen regio-nal economies. In the future, grass may be used for thesustainable production of chemicals, biomaterials and

0960-8524/$ - see front matter � 2007 Elsevier Ltd. All rights reserved.

doi:10.1016/j.biortech.2007.08.045

* Corresponding author. Tel.: +43 1 36006 6288; fax: +43 1 36006 6251.E-mail address: [email protected] (S. Novalin).

plant fibers. The Austrian Green Biorefinery is oneapproach towards the implementation of these objectives.The basic idea here is that, in analogy to concepts appliedin petrochemistry, the raw material grassland biomass isused with the whole plant to produce several productgroups without generating waste. As grassland biomassdoes not offer a specific major component, like sugar beet(sucrose) or corn (starch), it is compelling to establish amulti-product system (Biorefinery system). A key elementin the Austrian concept is the utilization of silage insteadof fresh biomass. During the fermentation process sugarsare converted into lactic acid and proteins are hydrolysedto free amino acids. Therefore, lactic acid and amino acidsare seen as the key compounds in such a Green Biorefinerysystem based on grass silage (Kromus et al., 2004).

Besides amino acids, interest in producing lactic acid isgrowing because of its great potential in the manufactureof biodegradable polymers (Datta et al., 1995). However,

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Nomenclature

BV bed volume–column volume for a packed col-umn

C0 initial concentration of lactic acid in startingsolution, g L�1

C* amount of lactic acid adsorbed by the resin,g g�1

CE lactic acid concentration at equilibrium, g L�1

Dinner inner diameter of column, cmH length of column, cmHPLC high performance liquid chromatographyk0A; k

0B capacity factors

LA lactic acidN theoretical plate numberR resolutionT temperature, �CV initial volume of starting solution, LV0 dead volume, mLVL maximum loaded volume, mLVfeed loaded volume, mLW weight of resin, ga relative retention

V.H. Thang, S. Novalin / Bioresource Technology 99 (2008) 4368–4379 4369

one main difficulty in lactic acid production lays on therecovery cost. To avoid the formation of gypsum generatedin the conventional recovery process, several new tech-niques have been developed for lactic acid recovery. Someof these techniques are electrodialysis (Heriban et al., 1993;Bailly, 2002), distillation with the simultaneous esterifica-tion reaction (Choi and Hong, 1999), ion exchange resin(Evangelista et al., 1994; Evangelista and Nikolov, 1996;Ponnampalam, 2001; Cao et al., 2002; Tong et al., 2004)and extraction (Matsumoto et al., 2003; Yankov et al.,2004).

In the case of grass silage juice, it seems somewhat diffi-cult to apply the above-mentioned techniques. Due to theheterogeneous nature of the silage microflora, the selectiveseparation of lactic acid requires delicate unit operations toproduce an end product of acceptable quality (Danneret al., 2000). Nevertheless, separation of lactic acid fromsilage juices was previously reported. It was shown that lac-tic acid with high content of impurities could be obtainedand therefore only low-grade applications such as animalfeed additive or road-de-icers were suggested (Danneret al., 2000; Madzingaidzo et al., 2002). Thang et al.(2004, 2005) reported that high-energy consumption wasrequired for removing ions other than lactate in lacticacid recovery from grass silage juice by two-stageelectrodialysis.

Chromatography is a powerful separation method thatwas developed initially for the extraction and purificationof complex mixtures of vegetal origin (Guiochon, 2002).It has been developed for many years as a very useful toolfor industrial applications. The use of this technique isincreasingly applied in the pharmaceutical industry, bio-technology as well as in the production of fine chemicals(Dechow, 1991). Several of the many advantages of chro-matography can be mentioned such as reduction of theeffluent volume and of the pollution load, avoidance ofchemical regenerants or a significant decrease of energyconsumption. The successful application of chromatogra-phy in sugar industry has inspired the researchers todevelop new chromatography systems for other applicationin industrial scale.

On the other hand, it should be noted that the resinsused for chromatographic separation in sugar industryseparation are ion exchange resin, which will be soonexhausted if the feed contains different ions in highamounts. It can be seen that chromatographic separationusing ion exchange resin as separation media will proba-bly not be practicable for the feed as silage juice. Althoughion exclusion chromatography has been successfullyapplied for separating components of molasses, namelyvery complex mixtures, it should be noted that the contentof different inorganic salts in silage juice is much higherthan that in molasses. Ion exchange chromatographywas also tested for lactic acid separation but the resinsrequired several regeneration steps and needed eluentsother than water such as acids, methanol, ammonia oreither their mixtures (Ponnampalam, 2001; Cao et al.,2002; Tong et al., 2004).

From the fact mentioned above, it goes to a new ideathat if a neutral resin is used, the regeneration step willbe much reduced. On the other hand, a simple and cheapeluent such as water will also be preferred to mentionedeluents. However, very few applications of chromatogra-phy using neutral resin were found for lactic acid separa-tion. Only one publication about using neutral polymericadsorbent for separation of citric acid from fermentationbroth was found (Kulprathipanja, 1988). Applications forAmberlite XAD1600 resin suggested in the data sheet fromRohm and Haas Company include recovery and purifica-tion of antibiotics, water-soluble steroids, amino acidsand proteins; removal of non-polar compounds from polarsolvents. There is, of course, no hint in the literature ofusing this resin for separation of lactic acid from a mixture.It is believed that this work is the first report about separa-tion of lactic acid using neutral polymeric resin. In thiswork, the recovery of undissociated lactic acid from grasssilage juice by chromatography using neutral adsorbentAmberlite XAD1600 was preliminarily investigated. Firstof all, the effects of operating parameters such as flow-rate,feed concentration and loaded volume were tested with themodel solutions. After that, the column separation withgrass silage juices was carried out.

4370 V.H. Thang, S. Novalin / Bioresource Technology 99 (2008) 4368–4379

2. Methods

2.1. Grass silage juice

The different silages, which were collected from differentfarms in the province of Styria, Austria, were mainly pro-duced from red clover (Trifolium pratense) and ryegrass(Lolium perenne). Silage juice was prepared by pressingensiled forages. Different silage juices were mixed andstored at �30 �C for further uses.

2.2. Chemicals

The resin used in experiments was polymeric adsorbentAmberlite XAD1600 (Rohm and Haas, Belgium). Theoriginal resin has water content of 63.8%. The resinwas then rinsed several times with high quality waterand then filtered by paper filter. The water-rinsed resin,which was used for experiments, has water content about65%.

The model solutions were prepared by mixing 50% –sodium lactate solution with glucose. The pH of obtainedsolutions was lowered to 2.0 by adding concentrated sulfu-ric acid solution. All reagents used in experiments were lab-oratory grade (Fluka, Austria).

Anion and cation standard solutions were purchasedfrom Fluka. Organic acid and sugar standard solutionswere, respectively, prepared from corresponding sub-stances (Fluka, Austria), which were all analytical grade.Amino acid standard solution was purchased from AlltechGROM GmbH, Germany.

2.3. Adsorption isotherms

Adsorption isotherms were determined by using a 1:10(w:v) ratio of resin and starting solution (Tong et al.,2004). Lactic acid used for adsorption isotherm was pur-chased from Merck, Germany. All other reagents were ofanalytical grade and were used without further purifica-tion. Aqueous solutions containing lactic acid, inorganicsalts (sodium sulfate and chloride), sugars (glucose andfructose) at different pHs were used as starting solutions.Finally, all vials containing the resin and starting solutionswere placed for 24 h in a water bath at 60 �C. Residual lac-tic acid concentrations in the liquid phase were measuredby HPLC and then, the amount of adsorbed lactic acidper gram of wet resin was calculated using the followingequation

C� ¼ ðC0 � CEÞVW

ð1Þ

where C* is amount of lactic acid adsorbed by the resin(g g�1); C0 is initial concentration of lactic acid in startingsolution (g L�1); CE is lactic acid concentration at equilib-rium (g L�1); V is initial volume of starting solution (L)and W is weight of wet resin (g).

2.4. Preparative chromatography

The laboratory scale preparative chromatography unitis made by Gold Fish GmbH, Austria. The jacketed100 · 2.5 cm column from Kronlab (Sinsheim, Germany)equipped with adjustable plungers was charged with resin.Resin was mixed with water treated by reverse osmosis for24 h at room temperature, and floating resins wereremoved. The resin bed was backwashed with reverseosmosis water to remove fine particles. The air trapped inthe resin was removed and the plunger was lowered tothe top of the resin bed. This step was followed by washingwith water until the pH at the outlet of column was similarto that of water.

After the preparation of column, chromatographic sep-aration was carried out. The feed solution was introducedinto the column via a sample loop or a peristaltic pump.A constant temperature circulator was used to maintainthe temperature of water in the jacket. Reverse osmosiswater was used as eluent, which was downwardly pumpedvia pump. Fractions were automatically collected by anautomatic sample collector and analyzed later. Conductiv-ity and pH of effluent were monitored online. After the sep-arations, a solution of NaOH (1 g L�1) was used to removethe possible impurities, which were still remained in thecolumn.

2.5. Determination of void volume

The void volume was determined by placing 10 ml of1 M NaCl solution at the top of the column, allowingmaterial to flow through the column at a fixed flow-rateof 2.04 cm min�1. Since NaCl has no interactions withthe resin, it will go through the column with the samevelocity of eluent. The signal was online measured by aconductivity meter and the time required for passing NaClthrough the column was recorded. From the obtained data,the void volume was calculated to be 320 ml.

2.6. Analyses

Concentration of organic acids was analyzed by HPLC(Merck–Hitachi) equipped with an Aminex HPX-87 H col-umn (Bio-Rad Co., USA) and RI detector. The columntemperature was maintained at 65 �C. The mobile phasewas 10 mM H2SO4 at flow-rate of 0.6 mL min�1.

Sugars were analyzed by HPLC (Dionex DX-500, USA)equipped with a CarboPac PA1 column at 25 �C and ED40Integrated Amperometric Detector using 25 mM NaOH asan eluent at flow-rate of 1 mL min�1.

Concentration of inorganic anions was analyzed usingion chromatography (Dionex DX-120, USA) equippedwith IonPac AS 14 (for anion analysis) and IonPac CS12A (for cation analysis) columns at 25 �C and a DS4Detection Stabilizer/Electrochemical Detector. The mobilephase for anion analysis was solution of NaHCO3/Na2CO3

(1 mM/3.5 mM) at flow-rate of 1.2 mL min�1. The mobile


phase for cation analysis was 20 mM methanesulfonic acidat flow-rate of 1.2 mL min�1.

Amino acids were analyzed by using HPLC (SummitHPLC Analytical System, Dionex, USA) with RF 2000Fluorescence Detector. The samples were analyzed afterprecolumn derivatisation with OPA (ortho-phthaldialde-hyde) using a GROM-SIL OPA-1 column (Alltech GROMGmbH, Germany). The column temperature was main-tained at 25 �C. The flow-rate was 1 mL min�1. Eluent Ais 25 mM sodium phosphate, pH 7.2/Tetrahydrofuran(995/5); Eluent B is 25 mM sodium phosphate, pH 7.2/MeOH/Acetonitrile (50/35/15). The analysis was carriedout by gradient elution: 100% A (0–2 min), 100–50% Aand 0–50% B (2–12 min), 50–40% A and 50–60% B (12–17 min), 40–0% A and 60–100% B (17–23 min), 100% B(23–28 min), 100% A (28–40 min).

0

0.2

0.4

0.6

0.8

1

0pH

C*,

g/g

of re

sin

87654321

Fig. 2. Effect of pH on the lactic acid adsorption in XAD1600 resin(T = 25 �C).

Y = 0.0013 XR2 = 0.9909

0.2

0.3

0.4

0.5

C*,

g/g

of re

sin

Lactic acid (experiment data)

Glucose

Inorganic salts

Model simulation

3. Results and discussion

3.1. Adsorption isotherm

Data sheet (Rohm and Hass Company, 2002) showsthat the neutral polymeric resin XAD1600 is non-ionicand hydrophobic adsorbent. Therefore this resin will selec-tively adsorb non-ionic species compared to ionic species.On the other hand, in aqueous solution, unionized lacticacid exists in equilibrium with lactate ions. It can be seenfrom Fig. 1 how pH affects on the equilibrium of the undis-sociated and dissociated acid forms. Therefore, the equilib-rium point of lactic acid dissociation can be shifted byvarying the concentration of hydrogen ion, namely pH ofsolution. Based o the lactic acid equilibrium and resinproperties, undissociated lactic acid will be separated fromthe other ionic species.

From the above comments, the sorption of lactic acid onan adsorbent, which will maximize the recovery of lacticacid, is expected to depend on the processing pH. The effectof pH on the lactic acid sorption of XAD1600 resin is pre-sented in Fig. 2. It is clear that the lower the pH of solu-tion, the greater the sorption of lactic acid. At pH lower

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

0 2 6

pH

Mol

e fr

actio

n

Lactate (L-)

pKa = 3.79

Lactic acid (HL)

1 543 87

Fig. 1. Disassociation curve of lactic acid at different pHs.

than pKa = 3.78, the lactic acid sorption strongly dependsupon pH. In contrast, this sorption changes slightly whenpH is higher than 4. In general, the sorption in resinsbelongs to two phenomena: adsorption and ionic exchange,which can happen simultaneously. Since the adsorbent inthis case is a neutral resin, the explanation for the sorptionof undissociated lactic acid is due to the strong affinity ofresin for relatively hydrophobic species. In other word, atlow pH, undissociated lactic acid is adsorbed on neutralresin.

An adsorption isotherm for lactic acid in mixture withinorganic salts and sugars is shown in Fig. 3. It is foundthat undissociated lactic acid was adsorbed on the resinwhereas the others such as inorganic salts and sugars were

0.0

0.1

0 50 100 150 200 250 300

CE, g.L-1

Fig. 3. Adsorption isotherm of lactic acid in the presence of impurities at60 �C, pH 2.0.

Table 1Composition of model feed solution

No. Component Concentration, g L�1

1 Lactic acid 260.062 Sulfate 152.453 Sodium 71.793 Glucose 76.23

0

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0 20 40 60 80 100Time, min

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cent

ratio

n, g

.L-1

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0 30 60 90 120 150

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cent

ratio

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.L-1

0

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0 15 30 45 60 75

Time, min

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cent

ratio

n, g

.L-1

Lactate Glucose

Sulfate Sodium

0

5

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15

20

25

30

0 10 20 30 40Time, min

Con

cent

ratio

n, g

.L-1

Lactate Glucose

Sulfate Sodium

a b

dc

Fig. 4. Chromatographic separation of non-lactic and lactic acid at different flow-rates. Conditions: column dimension = 2.5 · 100 cm (Dinner · H),temperature = 60 �C, Vfeed = 10 mL, flow-rate = 1.53 cm min�1 (a); 2.04 cm min�1 (b); 3.06 cm min�1 (c) and 6.12 cm min�1 (d).


not, thus lactic acid can be easily separated from sugarsand inorganic salts. The obtained data shows that lacticacid solution has a linear adsorption behavior on the resinand the data can be fitted well by the Henry isotherm. Sincethe adsorption isotherm was linear, there is nearly no lim-itation in mass adsorbed during the separation processwhen the concentration of lactic acid is up to 300 g L�1.

3.2. Effect of flow-rates on separation

Flow-rate is one of the most important parameter inprocess chromatography. In practice, the productivity is

Table 2Effect of flow-rate on lactic acid separation by chromatography

Flow-rate, cm min�1 Resolution, R Maximum concentrof lactic acid, g L�

1.53 1.18 22.062.04 1.16 23.003.06 1.09 22.896.12 0.95 16.85

a Productivity was calculated as sugar amount obtained per unit volume of

maximized by increasing the flow-rate to the highest possi-ble value (Felinger and Guiochon, 1992a, 1992b). Theeffects of different flow-rates on the separation performanceof non-lactic and lactic acid were investigated with modelsolution at four different levels: 1.53; 2.04; 3.06 and6.12 cm min�1 using the same feed. The composition offeed solution is shown in Table 1. The loaded volumewas 10 mL in all experiments.

The feed were pumped into the top of column, and thenreverse osmosis water was downwardly pumped into thecolumn as eluent. The eluted effluents were collected in15 ml fractions for later analysis off-line. It can be seen

ation1

Productivitya,kg m�3 h�1

Purity, % Yield, %

3.09 98.51 99.554.46 98.75 99.406.45 98.61 99.47

12.06 93.21 99.22

resin per unit time.

0

3

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0 20 40 60 80 100

0 20 40 60 80 100

0 20 40 60 80 100

Time, minC

once

ntra

tion,

g.L

-1

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cent

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n, g

.L-1

0

6

12

18

24

30

Time, min

Con

cent

ratio

n, g

.L-1

Lactate Glucose

Sulfate Sodium

c

b

a

Fig. 5. Effect of feed concentration on the separation of lactic acid.Conditions: temperature = 60 �C, flow-rate = 2.04 cm min�1, column:2.5 · 100 cm (Dinner · H); lactic acid concentration in feed = 78.29 g L�1

(a); 128.28 g L�1 (b); 260.06 g L�1 (c).

Table 3Composition of feed solutions

Feed Lactic acid,g L�1

Glucose,g L�1

Sodium,g L�1

Sulfate,g L�1

1 260.06 76.23 71.79 152.452 128.78 38.74 35.55 76.973 78.29 23.29 20.22 44.42


from Fig. 4 that that in all experiments, the curves havesimilar profiles and the volume retentions for lactic acidwere almost not changed (1.21–1.33 BV) but the base ofthe peaks was broader. This will cause a decreasing the res-olution between sugar and non-sugar peaks. Data in Table2 shows that the resolution R decreased from 1.18 to 0.95with the increasing flow-rate. At low flow-rates of 1.53and 2.04 cm min�1, the difference between the resolutionswas very slightly but the productivity was considerable dif-ference. When the flow-rate increased to 6.12 cm min�1 theresolution decreased significantly. However, in processchromatography the resolution is not always the primor-dial factor (Stead, 1998). For economical reason, the pro-ductivity is firstly considered.

As mentioned above, the productivity increased nearlylinearly with the increase of flow-rate. However, it shouldbe noted that high flow-rates mean high-pressure dropand significant dilution. In this work, although the pressuredrops were lower than 3 bar, which was still far from thelimit for system, the dilution of lactic acid fraction showedno interest in further increasing flow-rate. It can beobserved in Fig. 4 that the highest concentration of lacticacid was similar when flow-rate was varying from 1.53 to3.06 cm min�1 but decreased considerably when flow-rateincreased to 6.12 cm min�1. The data in Table 2 shows thatat flow-rates varying from 1.53 to 3.06 cm min�1, it seemsthat the flow-rate had minor effect on the purity. With sim-ilar recovery yields, the purity of lactic acid fractions wasalso similar (around 98.5%). Although it was expected thatthe purity would decrease with the increase of flow-rate thepurity of lactic acid fractions in this case was almost simi-lar. The purity decreased significantly to 93.21%, onlywhen flow-rate was relatively high (6.12 cm min�1).

3.3. Effect of feed concentration on separation

Preparative separations are carried out at high concen-tration under such conditions that the equilibrium iso-therms of the feed components in the chromatographicsystem are non-linear (Felinger and Guiochon, 1996b).However, in this work, the feed concentrations used werestill in linear area. The effects of feed concentration onthe separation performance of non-lactic and lactic acidwere investigated with model solution at three different lev-els using the same feed volume of 10 mL. The obtainedresults in previous part showed that there was almost nodifference in resolution at flow-rates of 1.53 and2.04 cm min�1, but there was a significant difference in pro-ductivity. Therefore, the flow-rate of 2.04 cm min�1 was

used in further experiments. The composition of feed solu-tion is shown in Table 3.

Fig. 5 shows the effect of feed concentration on the sep-aration performance. Like the case of different flow-rates,in this case the curves have similar profiles and the volumeretentions for lactic acid (1.21–1.35 BV). With the increase

Table 4Effect of feed concentration on lactic acid separation by chromatography

LA concentrationin feed, g L�1

Maximum concentrationof LA, g L�1

Resolution, R Productivitya,kg m�3 h�1

Purity, % Yield, %

260.06 23.00 1.16 4.46 98.75 99.40128.78 11.27 1.32 2.23 99.35 99.7978.29 6.47 1.45 1.29 99.86 99.57

a Productivity was calculated as sugar amount obtained per unit volume of resin per unit time. LA means lactic acid.


of feed concentration, the maximum concentration of lacticacid increased respectively. However, in all cases the ratiobetween lactic acid concentration in feed and the maximumconcentration of lactic acid was similar.

It can be seen from Table 4 that the low concentrationof feed can improve the purity. With similar recoveryyields, the purity increased from 98.75% to 99.86% whenthe feed concentration decreased from 260.06 g L�1 to78.29 g L�1, respectively. Similarly to the purity, the reso-lution increased significantly with the decrease of feed con-centration. In contrast, the most important parameter forpractical operation, namely the productivity, decreasedproportionally with the decreasing feed concentration.

3.4. Effect of loaded volumes on separation

In production scale, the objective is to obtain a maxi-mum yield with a given degree of separation. The sampleshould be made as large as possible within the limits setby the required separation. The sample volume could beas large as the separation volume; however, due to non-equilibrium between the mobile phase and longitudinal dif-fusion in the band, the zones will always be broadened. Thesample volume must therefore be smaller that the separa-tion volume. In process chromatography, the feed volumeup to 20% of the bed volume can be applied (Stead,1998). In this work, the loaded volume was investigatedwith model solutions at three different levels: 0.02, 0.04and 0.10 BV. It can be seen from Fig. 6 that the retentiontime for lactic acid was shorter when increasing the loadedvolume. In the same time, there was a considerable degreeof overlap of non-lactic and lactic acid peaks. This overlapzone is very important for the recovery yield and the purity.

Table 5 shows details of effects of loaded volume on sep-aration performance. The resolution decreased remark-ably, from 1.16 to 0.55 when increasing the loadedvolume from 2.2% to 10% of column volume. The maxi-mum concentration of lactic acid increased nearly propor-tionally to the loaded volume. When the loaded volumewas 2.22% of the bed volume, the difference in concentra-tion between feed and the maximum concentration wasthe factor of 11.3. This factor was reduced to only 2.4 whenthe loaded volume reached to 10% of the bed volume. Notonly the maximum concentration of lactic acid, but also theproductivity was considerably improved by increasing theloaded volume. However, with the similar recovery yields,the purity decreased significantly with the increase of

loaded volume. The purity of lactic acid was very poor(92.93%) when the loaded volume was 10% of bed volume.

As mentioned above, because of the economical reason,the most important parameter in process chromatographyis productivity. However, the recovery yield has to be takeninto consideration. In practice, a recovery yield about 90%is usually considered as satisfactory (Felinger and Guio-chon, 1996a). Table 6 shows the relation between the recov-ery yield and other separation parameters. The decrease ofrecovery yield can improve the purity but not so much.When the recovery yield decreased from 99.50% to86.77%, the purity increased only from 92.93% to96.71%. It can be found that a 1% reduction of the recoveryyield resulted in about 1% reduction of the productivity.On the other hand, if the recovery yield is lower, the dilu-tion factor as is more improved. The concentration of lacticacid in final solution (a mixture of collected fractions)increased from 36.20 to 63.14 g L�1 when the recoveryyield decreased from 99.50% to 86.77%. In practice, thedilution of the product is important because concentratedsolutions are required for the commercial products, andtherefore, a lot of energy has to put in concentration step.

3.5. Lactic acid separation from silage juice

3.5.1. Pulse test for separation of lactic acid from silage juice

The column separation was tested in order to determinethe ability of separating lactic acid from a complex mixtureby resin XAD1600. The feed used for pulse test was thepermeate of nanofiltration using membrane MPF-36. Thefeed volume was 10 mL and the feed composition is pre-sented in Table 7.

Fig. 7 presents elution profile of lactic acid separationfrom silage juice by chromatography. The curve of conduc-tivity shows clearly two separated peaks where the firstpeak was much higher than the second one. It proved thatthe salts were separated from the others. The low conduc-tivity of the second peak proved that almost lactic acid wasin undissociated form. This observation agreed with thechange of pH. Firstly, the pH of effluent decreased togetherwith the increase of inorganic salts because mineral acid,which was used to adjust pH of feed solution, was elutedtogether with inorganic salts and sugars. After salt fraction,the pH of effluent increased slightly, from 1.8 to 3.0, butstill lower than pKa of lactic acid. This also showed thatlactic acid obtained was in undissociated form.

It can be seen in Fig. 7 that, after about 1.7 BV of water,nearly all lactic acid can be easily eluted from loaded


column. The data shows that lactic acid was satisfactoryseparated from inorganic salts and sugars, but there wasno separation between sugars and inorganic salts and thesecompounds were eluted together in the first fraction. The

0

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100

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cent

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.L-1

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90

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Time, min

Con

cent

ratio

n, g

.L-1

Lactate Glucose

Sulfate Sodium

0 20 40 60 80

0 20 40 60 80 100

a

b

c

Fig. 6. Effect of loaded volume on lactic acid separation from modelsolutions. Conditions: temperature = 60 �C, flow-rate = 2.04 cm min�1,column: 2.5 · 100 cm (Dinner · H); Vfeed = 10 mL (a); 20 mL (b); 45 mL(c).

resolution R between lactic acid and inorganic salts was1.63, higher than the highest resolution (R = 1.45) in exper-iments with model solutions. The high resolution wasexplained by lower concentration of lactic acid in silagejuice when comparing with model solutions. It is knownthat when the resolution R is 1.5, more than 99% sub-stances are separated. This agreed with the result in adsorp-tion isotherm of salts and sugars on the resin. However,unlike the model solutions, silage juice contains also aceticacid and the purity of lactic acid obtained was affected bythis component. Although acetic acid was washed out laterthan lactic acid, it can be seen that acetic acid was not prac-tically separated from lactic acid.

3.5.2. Preparative separation of lactic acid fromconcentrated silage juice

Unlike analytical chromatography, in which the aim isto separate all individual components of a mixture ascompletely as possible with subsequent identification ofthe peaks, in process chromatography the aim of separa-tion often involves detailed economical–chemical optimi-zation calculations. Due to the column dimensions, costsfor solvents and packing or pre-packed columns becomemore and more important with increasing column diame-ters. For maximizing productivity, process chromatogra-phy columns are often overloaded. According to generalunderstanding, industrial scale chromatography is gener-ally carried out under mass and/or volume overloadedconditions in order to increase the product productivity.In volume overloading, the sample concentration is main-tained in the linear region of the isotherm and the volumeis increased until the productivity is optimized. In massoverloading, the sample concentration is increased beyondthe linear adsorption region, resulting in asymmetric bandprofiles. A combination of volume and mass overloadingis commonly used to maximize productivity in preparativeelution chromatography. However, a concentration over-load is only possible, if the solubility of the substancesin the feed solution is large enough. In the case of molas-ses separation, the feed concentration is from 60% to 80%(w/w). However, one important factor, which limits theincrease of concentration, is solubility of substances infeed. In this work, silage juice was concentrated untillactate concentration reached about 140 g L�1. Thisconcentration was considered the maximum concentra-tion of feed solution since further concentration stepmight bring difficulties, such as high viscosity of solutionor crystallization of some substances. The composition offeed mixture is shown in Table 8. Interestingly, aceticacid concentration in concentrated silage juice was similarto that in silage juice. The reason was due to the evapo-ration of this compound during concentration process ofjuice.

The aim of this work is not optimizing the separationprocess, therefore the loaded volume was used based onthe volume loadability. The volume loadability can be cal-culated by formula (Scott and Kucera, 1976):

Table 5Effect of loaded feed volume on lactic acid separation by chromatography

Loaded volume,% of bed volume

Maximum concentrationof lactic acid, g L�1

Resolution, R Productivitya,kg m�3 h�1

Purity, % Yield, %

2.22 23.00 1.16 4.46 98.75 99.404.44 48.52 0.90 8.93 97.90 99.41

10.00 106.38 0.55 20.29 92.93 99.50

a Productivity was calculated as sugar amount obtained per unit volume of resin per unit time.


V L ¼ V 0 ða� 1Þk0A �2ffiffiffiffiNp ð2þ k0A þ k0BÞ

� �ð4:2Þ

Table 6Effect of recovery yield on separation performance

Yield,%

Purity,%

Productivity 1,kg m�3 h�1

Lactic acidconcentration, g L�1

Dilutionfactor

99.50 92.93 20.29 36.20 7.295.36 96.99 19.45 40.82 6.492.54 96.90 18.77 51.80 5.086.77 96.71 17.69 63.14 4.1

Table 7Composition of silage juice used as feed for pulse test

Substance Concentration,g L�1

Lactic acid 35Acetic acid 2.0Amino acids 22.8Sugars (glucose, fructose, sucrose, arabinose,

xylose, . . .)26.85

Inorganic salts (K+, Na+, Ca2+, Mg2+, NHþ4 , Cl�,NO�3 ;SO2�

4 )62.78

0

2

4

6

8

10

0.0 0.5 1.0 1.5 2.0 2.5

Bed volume

Con

cent

ratio

n, g

.L-1

0

2

4

6

8

10

12

14

16

pHC

ondu

ctiv

ity, m

S.cm

-1

Inorganic salts Sugars Lactic acid

Acetic acid pH Conductivity

Fig. 7. Elution profile of lactic acid separation from silage juice bychromatography. Conditions: Vfeed = 10 ml, flow-rate = 2.04 cm min�1,T = 60 �C, column: 2.5 · 100 cm (Dinner · H).

where VL is the maximum load volume; V0 is the dead vol-ume; a is relative retention; k0A and k0B are capacity factorsand N is the theoretical plate number.

As can be seen from the formula (4.2), the volume loa-dability linearly depends on the dead volume of the columnused, and otherwise it only depends on the k 0 values of thecomponents to be separated and on the separation effi-ciency of the column. From obtained data in pulse tests,the volume loadability was calculated as about 40 mL forabove column.

Fig. 8 presents elution profile of lactic acid separationfrom 4-fold concentrated silage juice by chromatography.It was found that the behavior of conductivity was not

0

20

40

60

80

100

120

0.0 0.5 1.0 1.5 2.0 2.5Bed volume

Con

duct

ivity

, mS.

cm-1

Con

cent

ratio

n, g

.L-1

0

2

4

6

8

10

pH

Sugars Lactic acid Acetic acid

Inorganic salts Conductivity pH

Fig. 8. Separation of lactic acid from concentrated silage juice bypreparative chromatography. Conditions: Vfeed = 40 ml, flow-rate = 2.04cm min�1, T = 60 �C, column: 2.5 · 100 cm (Dinner · H).

Table 8Composition of concentrated silage juice as feed for chromatographicseparation

Substance Concentration,g L�1

Lactic acid 140.0Acetic acid 2.0Amino acids 90.0Sugars (glucose, fructose, sucrose, arabinose,

xylose, . . .)105.2

Inorganic salts (K+, Na+, Ca2+, Mg2+, NHþ4 , Cl�,NO�3 ; SO2�

4 )249.1


similar to that in pulse test experiment: only one peak wasobserved instead of two. The lactic acid peak could not beseen based on the curve of conductivity. These resultsproved that the higher volume is loaded, the bigger overlaparea will be resulted. Therefore, inorganic salts in the over-lap area caused the conductivity higher than that caused byundissociated lactic acid. Like experiments with modelsolutions, lactic acid was completely eluted after 1.7 BVof eluent. Although the resolution between lactic acidand inorganic salts slightly decreased in comparison withthe result obtained in pulse test, the separation betweenthese compounds was still very good. However, the prob-lem in separating lactic acid from acetic was still remained.It can be seen in Fig. 8 that acetic acid was eluted nearlytogether with lactic acid. Unfortunately, due to the verycomplex composition, silage juice contains not only aceticacid but also other organic acids with smaller amounts.These organic acids caused the same problem in separatinglactic acid from other organic acids.

Besides acetic acid, amino acids also occurred in lacticacid fraction as impurities. Fig. 9 shows that amino acidsoccurred in all collected fractions. In lactic acid fraction,the main remaining amino acids were valine, leucine, iso-leucine and lysine.

After the separation, two main fractions were obtained:lactic and non-lactic fraction. Lactic acid fraction has con-centration of 17.94 g L�1 with the recovery yield of 97%and purity of 94%. More than 99% of inorganic salts andsugars were separated from lactic acid. The compositionof lactic acid fraction obtained after separation processwas analyzed in HPLC. The result is presented in Fig. 10,where besides lactic acid and acetic acid other organic acidssuch as citric and succinic acids are existed. Besides thepurity problem, another disadvantage was the dilution ofproduct. In this investigation, the dilution factor was about7.9. However, it was quite difficult to improve the dilutionfactor. It seems that the loaded volume was close to thelimitation for column separation. Moreover, unlike thecase of model solutions, the concentrated silage juice hasa limitation in maximum lactic acid concentration.

0

4

8

12

16

20

0.0 0.5 1.0 1.5 2Bed volume

Con

cent

ratio

n, g

.L-1

Fig. 9. Effluent profiles of amino acids for XAD1600 resin. Conditions:2.5 · 100 cm (Dinner · H).

3.5.3. Proposed processes for separation of lactic acid from

silage juice

From the obtained results, a possible process for sepa-rating lactic acid from grass silage juice is proposed asFig. 11. Silage juice has to be concentrated before beingfurther processed. The solubility of concentrated solutionshould be taken into account. A concentration factor offour of fivefolds is suggested. This concentration stepcan also reduce a great part of acetic acid, thus minimizeits amount in the lactic acid product. The acidificationstep is done by using mineral acids, but sulfuric acid ispreferred. When concentrated sulfuric acid is used foracidifying the juice, the feed for chromatography will beless diluted. Other advantages of using sulfuric acid arethat it is cheaper and less corrosive than other mineralacids. The acidified juice is then applied to a chromatogra-phy column containing neutral polymeric resin likeXAD1600. Lactic acid was then eluted from the columnby using water that is condensed from evaporation steps.However, if lactic acid is produced like this process, a partof amino acids will be still remained as impurities. For theresin, it was observed that colored components wereadsorbed into the resin. Although in this work, this phe-nomenon seems not affect the performance of lactic acidseparation, the more detailed investigation about the effectof a full-color-adsorbed column on the lactic acid separa-tion is needed.

The problem of amino acid as impurity could be mini-mized by using chromatography in combination with elec-trodialysis. Thang et al. (2004) reported that about 90% ofamino acids were separated from lactate fraction by elec-trodialysis. Thus, an alternative process, which combineschromatography and electrodialysis could be proposed(Fig. 11). Firstly, amino acids will be separated by electro-dialysis because at neutral pH, most of amino acids are inneutral forms and therefore they will be remained in thediluate of electrodialysis. The concentrate of electrodialysiscontaining mainly charged components such as lactic acidand inorganic salts will further be processed like proposedscheme in Fig. 11.

.0 2.5

Group 1

Group 2

Group 3

Group 1: Aspartate, Glutamate, Asparagine, Glutamine, Histidine, Glycine, Threonine, Arginine, Alanine, Gaba.

Group 2: Valine, Methionine. Group 3: Tyrosine, Leucine,

Isoleucine, Lysine.

Vfeed = 40 ml, flow-rate = 2.04 cm min�1, temperature = 60 �C, column:

Fig. 11. Scheme for lactic acid separation and production from grasssilage juice.

1 - C

itrat

e

2 -

Glu

cose

3 - F

ruct

ose

4 - S

ucci

nate

5 - L

acta

te

6 - A

ceta

teFig. 10. HPLC chromatogram of the lactic acid separated from grasssilage juice.


4. Conclusions

This work has shown that the chromatographic separa-tion using neutral adsorbent resins like AmberliteXAD1600 could simply solve the desalting problem inrecovery of lactic acid from grass silage juice. More than99% of inorganic salts as well as sugars were removed withrecovery yields over 99.4%.

However, due to the complex composition of the grasssilage juices, the purity of lactic acid decreased to 94% atthe recovery yield of 97%. Although about 99% of inor-

ganic salts and sugars were separated from lactic acid,organic acids in general and acetic acid in particular causeda purity problem. Besides the organic acids, some aminoacids were remained in the products as impurities. Forthe production of lactic acid from raw materials having lesspurity, the above-mentioned method should further beinvestigated more in detail. However, the results from thiswork can show a good hint for a single-step separation oflactic acid produced from quite pure raw materials, such asglucose syrup or starchy hydrolysates. In these cases, thefermentation broths will contain much less free amino acidsand organic acids. Finally, due to the dilution of product,more energy consumption will be needed for concentrationstep to obtain commercial product.

Acknowledgements

This research has been carried out in connection withthe programme ‘‘Fabrik der Zukunft’’ of the Austrian Fed-eral Ministry of Traffic, Innovation and Technology (Pro-ject No.806097). The authors would like to thank OAD(Osterreichischer Austauschsdienst/Austrian Academic Ex-change Service) for granting V.H. Thang a doctoralscholarship.

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