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Bioresource Technology 99 (2008) 43684379

Green Biorenery: Separation of lactic acid from grass silage juice by chromatography using neutral polymeric resin
Vu Hong Thang
a

a,b

, Senad Novalin

a,*

Department of Food Science and Technology, Vienna University of Natural Resources and Applied Life Sciences (BOKU), Muthgasse 18, A-1190 Vienna, Austria b 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 2007 Available 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 separation using neutral polymeric resin Amberlite XAD1600 was investigated. Up to now, there is no hint in the literatures about using neutral polymeric resin for lactic acid separation from a mixture. Important factors (ow-rate, concentration of feed and loaded volume) that aect separation performance were rstly investigated with model solutions. The obtained results showed that lactic acid solutions with 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 were carried out. Due to the complex composition of the feed, the purity of products decreased to 94% at a recovery yield of 97%. Although 99% of inorganic salts and sugars were separated from lactic acid organic acids in general and acetic acid in particular caused a purity problem. It seems that organic acids could not be separated from lactic acid by neutral resin Amberlite XAD1600. Besides the organic acid 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 currently undergoing structural changes, which are characterized by a decrease of livestock and dairy farming. One of the consequences is an increase of unused grassland biomass (grass) and uncultivated grassland (fallow land), respectively. It was estimated that, in the medium term, 750,000 tons of dry matter per year would be available from grassland in Austria (Forschungsforum BMVIT, 2004). Innovative technology concepts for the utilization of this unused grass can provide the raw material for the manufacture of future-oriented products, and thus strengthen regional economies. In the future, grass may be used for the sustainable production of chemicals, biomaterials and
*

Corresponding author. Tel.: +43 1 36006 6288; fax: +43 1 36006 6251. E-mail address: Senad.Novalin@boku.ac.at (S. Novalin).

plant bers. The Austrian Green Biorenery is one approach towards the implementation of these objectives. The basic idea here is that, in analogy to concepts applied in petrochemistry, the raw material grassland biomass is used with the whole plant to produce several product groups without generating waste. As grassland biomass does not oer a specic major component, like sugar beet (sucrose) or corn (starch), it is compelling to establish a multi-product system (Biorenery system). A key element in the Austrian concept is the utilization of silage instead of fresh biomass. During the fermentation process sugars are converted into lactic acid and proteins are hydrolysed to free amino acids. Therefore, lactic acid and amino acids are seen as the key compounds in such a Green Biorenery system based on grass silage (Kromus et al., 2004). Besides amino acids, interest in producing lactic acid is growing because of its great potential in the manufacture of biodegradable polymers (Datta et al., 1995). However,

0960-8524/$ - see front matter 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2007.08.045

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Nomenclature BV bed volumecolumn volume for a packed column C0 initial concentration of lactic acid in starting solution, g L1 * C amount of lactic acid adsorbed by the resin, g g1 CE lactic acid concentration at equilibrium, g L1 Dinner inner diameter of column, cm H length of column, cm HPLC high performance liquid chromatography k 0A ; k 0B capacity factors LA N R T V V0 VL Vfeed W a lactic acid theoretical plate number resolution temperature, C initial volume of starting solution, L dead volume, mL maximum loaded volume, mL loaded volume, mL weight of resin, g relative retention

one main diculty in lactic acid production lays on the recovery cost. To avoid the formation of gypsum generated in the conventional recovery process, several new techniques have been developed for lactic acid recovery. Some of these techniques are electrodialysis (Heriban et al., 1993; Bailly, 2002), distillation with the simultaneous esterication 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 dicult to apply the above-mentioned techniques. Due to the heterogeneous nature of the silage microora, the selective separation of lactic acid requires delicate unit operations to produce an end product of acceptable quality (Danner et al., 2000). Nevertheless, separation of lactic acid from silage juices was previously reported. It was shown that lactic acid with high content of impurities could be obtained and therefore only low-grade applications such as animal feed additive or road-de-icers were suggested (Danner et al., 2000; Madzingaidzo et al., 2002). Thang et al. (2004, 2005) reported that high-energy consumption was required for removing ions other than lactate in lactic acid recovery from grass silage juice by two-stage electrodialysis. Chromatography is a powerful separation method that was developed initially for the extraction and purication of complex mixtures of vegetal origin (Guiochon, 2002). It has been developed for many years as a very useful tool for industrial applications. The use of this technique is increasingly applied in the pharmaceutical industry, biotechnology as well as in the production of ne chemicals (Dechow, 1991). Several of the many advantages of chromatography can be mentioned such as reduction of the euent volume and of the pollution load, avoidance of chemical regenerants or a signicant decrease of energy consumption. The successful application of chromatography in sugar industry has inspired the researchers to develop new chromatography systems for other application in industrial scale.

On the other hand, it should be noted that the resins used for chromatographic separation in sugar industry separation are ion exchange resin, which will be soon exhausted if the feed contains dierent ions in high amounts. It can be seen that chromatographic separation using ion exchange resin as separation media will probably not be practicable for the feed as silage juice. Although ion exclusion chromatography has been successfully applied for separating components of molasses, namely very complex mixtures, it should be noted that the content of dierent inorganic salts in silage juice is much higher than that in molasses. Ion exchange chromatography was also tested for lactic acid separation but the resins required several regeneration steps and needed eluents other than water such as acids, methanol, ammonia or either their mixtures (Ponnampalam, 2001; Cao et al., 2002; Tong et al., 2004). From the fact mentioned above, it goes to a new idea that if a neutral resin is used, the regeneration step will be much reduced. On the other hand, a simple and cheap eluent such as water will also be preferred to mentioned eluents. However, very few applications of chromatography using neutral resin were found for lactic acid separation. Only one publication about using neutral polymeric adsorbent for separation of citric acid from fermentation broth was found (Kulprathipanja, 1988). Applications for Amberlite XAD1600 resin suggested in the data sheet from Rohm and Haas Company include recovery and purication of antibiotics, water-soluble steroids, amino acids and proteins; removal of non-polar compounds from polar solvents. There is, of course, no hint in the literature of using this resin for separation of lactic acid from a mixture. It is believed that this work is the rst report about separation of lactic acid using neutral polymeric resin. In this work, the recovery of undissociated lactic acid from grass silage juice by chromatography using neutral adsorbent Amberlite XAD1600 was preliminarily investigated. First of all, the eects of operating parameters such as ow-rate, feed concentration and loaded volume were tested with the model solutions. After that, the column separation with grass silage juices was carried out.

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2. Methods 2.1. Grass silage juice The dierent silages, which were collected from dierent farms in the province of Styria, Austria, were mainly produced from red clover (Trifolium pratense) and ryegrass (Lolium perenne). Silage juice was prepared by pressing ensiled forages. Dierent silage juices were mixed and stored at 30 C for further uses. 2.2. Chemicals The resin used in experiments was polymeric adsorbent Amberlite XAD1600 (Rohm and Haas, Belgium). The original resin has water content of 63.8%. The resin was then rinsed several times with high quality water and then ltered by paper lter. The water-rinsed resin, which was used for experiments, has water content about 65%. The model solutions were prepared by mixing 50% sodium lactate solution with glucose. The pH of obtained solutions was lowered to 2.0 by adding concentrated sulfuric acid solution. All reagents used in experiments were laboratory grade (Fluka, Austria). Anion and cation standard solutions were purchased from Fluka. Organic acid and sugar standard solutions were, respectively, prepared from corresponding substances (Fluka, Austria), which were all analytical grade. Amino acid standard solution was purchased from Alltech GROM 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 purchased from Merck, Germany. All other reagents were of analytical grade and were used without further purication. Aqueous solutions containing lactic acid, inorganic salts (sodium sulfate and chloride), sugars (glucose and fructose) at dierent pHs were used as starting solutions. Finally, all vials containing the resin and starting solutions were placed for 24 h in a water bath at 60 C. Residual lactic acid concentrations in the liquid phase were measured by HPLC and then, the amount of adsorbed lactic acid per gram of wet resin was calculated using the following equation C C 0 C E V W 1

2.4. Preparative chromatography The laboratory scale preparative chromatography unit is made by Gold Fish GmbH, Austria. The jacketed 100 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 for 24 h at room temperature, and oating resins were removed. The resin bed was backwashed with reverse osmosis water to remove ne particles. The air trapped in the resin was removed and the plunger was lowered to the top of the resin bed. This step was followed by washing with water until the pH at the outlet of column was similar to that of water. After the preparation of column, chromatographic separation was carried out. The feed solution was introduced into the column via a sample loop or a peristaltic pump. A constant temperature circulator was used to maintain the temperature of water in the jacket. Reverse osmosis water was used as eluent, which was downwardly pumped via pump. Fractions were automatically collected by an automatic sample collector and analyzed later. Conductivity and pH of euent were monitored online. After the separations, a solution of NaOH (1 g L1) was used to remove the possible impurities, which were still remained in the column. 2.5. Determination of void volume The void volume was determined by placing 10 ml of 1 M NaCl solution at the top of the column, allowing material to ow through the column at a xed ow-rate of 2.04 cm min1. Since NaCl has no interactions with the resin, it will go through the column with the same velocity of eluent. The signal was online measured by a conductivity meter and the time required for passing NaCl through 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 (MerckHitachi) equipped with an Aminex HPX-87 H column (Bio-Rad Co., USA) and RI detector. The column temperature was maintained at 65 C. The mobile phase was 10 mM H2SO4 at ow-rate of 0.6 mL min1. Sugars were analyzed by HPLC (Dionex DX-500, USA) equipped with a CarboPac PA1 column at 25 C and ED40 Integrated Amperometric Detector using 25 mM NaOH as an eluent at ow-rate of 1 mL min1. Concentration of inorganic anions was analyzed using ion chromatography (Dionex DX-120, USA) equipped with IonPac AS 14 (for anion analysis) and IonPac CS 12A (for cation analysis) columns at 25 C and a DS4 Detection Stabilizer/Electrochemical Detector. The mobile phase for anion analysis was solution of NaHCO3/Na2CO3 (1 mM/3.5 mM) at ow-rate of 1.2 mL min1. The mobile

where C* is amount of lactic acid adsorbed by the resin (g g1); C0 is initial concentration of lactic acid in starting solution (g L1); CE is lactic acid concentration at equilibrium (g L1); V is initial volume of starting solution (L) and W is weight of wet resin (g).

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phase for cation analysis was 20 mM methanesulfonic acid at ow-rate of 1.2 mL min1. Amino acids were analyzed by using HPLC (Summit HPLC Analytical System, Dionex, USA) with RF 2000 Fluorescence Detector. The samples were analyzed after precolumn derivatisation with OPA (ortho-phthaldialdehyde) using a GROM-SIL OPA-1 column (Alltech GROM GmbH, Germany). The column temperature was maintained at 25 C. The ow-rate was 1 mL min1. Eluent A is 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 carried out by gradient elution: 100% A (02 min), 10050% A and 050% B (212 min), 5040% A and 5060% B (12 17 min), 400% A and 60100% B (1723 min), 100% B (2328 min), 100% A (2840 min).

than pKa = 3.78, the lactic acid sorption strongly depends upon pH. In contrast, this sorption changes slightly when pH is higher than 4. In general, the sorption in resins belongs to two phenomena: adsorption and ionic exchange, which can happen simultaneously. Since the adsorbent in this case is a neutral resin, the explanation for the sorption of undissociated lactic acid is due to the strong anity of resin for relatively hydrophobic species. In other word, at low pH, undissociated lactic acid is adsorbed on neutral resin. An adsorption isotherm for lactic acid in mixture with inorganic salts and sugars is shown in Fig. 3. It is found that undissociated lactic acid was adsorbed on the resin whereas the others such as inorganic salts and sugars were
1

3. Results and discussion

C*, g/g of resin

0.8

3.1. Adsorption isotherm Data sheet (Rohm and Hass Company, 2002) shows that the neutral polymeric resin XAD1600 is non-ionic and hydrophobic adsorbent. Therefore this resin will selectively adsorb non-ionic species compared to ionic species. On the other hand, in aqueous solution, unionized lactic acid exists in equilibrium with lactate ions. It can be seen from Fig. 1 how pH aects on the equilibrium of the undissociated and dissociated acid forms. Therefore, the equilibrium point of lactic acid dissociation can be shifted by varying the concentration of hydrogen ion, namely pH of solution. Based o the lactic acid equilibrium and resin properties, undissociated lactic acid will be separated from the other ionic species. From the above comments, the sorption of lactic acid on an adsorbent, which will maximize the recovery of lactic acid, is expected to depend on the processing pH. The eect of pH on the lactic acid sorption of XAD1600 resin is presented in Fig. 2. It is clear that the lower the pH of solution, the greater the sorption of lactic acid. At pH lower
1.0 0.9 0.8 0.7 Lactate (L-)

0.6

0.4

0.2

0 0 1 2 3 4 5 6 7 8

pH

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

0.5 Lactic acid (experiment data) 0.4 Glucose Inorganic salts 0.3 Model simulation

C*, g/g of resin

0.2

Y = 0.0013 X R2 = 0.9909

0.1

0.0 0 50 100 150 200 250 300

CE, g.L-1

Mole fraction

0.6 0.5 0.4 0.3 0.2 0.1 0.0 0

Lactic acid (HL) pKa = 3.79

Fig. 3. Adsorption isotherm of lactic acid in the presence of impurities at 60 C, pH 2.0.

Table 1 Composition of model feed solution No.

1 2 3 4 5 6 7 8

Component Lactic acid Sulfate Sodium Glucose

Concentration, g L1 260.06 152.45 71.79 76.23

pH

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

1 2 3 3

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a
Concentration, g.L-1

30

b
Concentration, g.L-1
0 30 60 90 120 150

30

25 20

25 20

15 10

15

10 5

5 0

0 0 20 40 60 80 100

Time, min

Time, min

c
Concentration, g.L-1

30

d
Concentration, g.L-1

30

25

25

20

20

15

15

10

10

0 0 15 30 45 60 75

0 0 10 20 30 40

Time, min
Lactate Sulfate Glucose Sodium Lactate Sulfate

Time, min
Glucose Sodium

Fig. 4. Chromatographic separation of non-lactic and lactic acid at dierent ow-rates. Conditions: column dimension = 2.5 100 cm (Dinner H), temperature = 60 C, Vfeed = 10 mL, ow-rate = 1.53 cm min1 (a); 2.04 cm min1 (b); 3.06 cm min1 (c) and 6.12 cm min1 (d).

not, thus lactic acid can be easily separated from sugars and inorganic salts. The obtained data shows that lactic acid solution has a linear adsorption behavior on the resin and the data can be tted well by the Henry isotherm. Since the adsorption isotherm was linear, there is nearly no limitation in mass adsorbed during the separation process when the concentration of lactic acid is up to 300 g L1. 3.2. Eect of ow-rates on separation Flow-rate is one of the most important parameter in process chromatography. In practice, the productivity is
Table 2 Eect of ow-rate on lactic acid separation by chromatography Flow-rate, cm min1 1.53 2.04 3.06 6.12
a

maximized by increasing the ow-rate to the highest possible value (Felinger and Guiochon, 1992a, 1992b). The eects of dierent ow-rates on the separation performance of non-lactic and lactic acid were investigated with model solution at four dierent levels: 1.53; 2.04; 3.06 and 6.12 cm min1 using the same feed. The composition of feed solution is shown in Table 1. The loaded volume was 10 mL in all experiments. The feed were pumped into the top of column, and then reverse osmosis water was downwardly pumped into the column as eluent. The eluted euents were collected in 15 ml fractions for later analysis o-line. It can be seen

Resolution, R 1.18 1.16 1.09 0.95

Maximum concentration of lactic acid, g L1 22.06 23.00 22.89 16.85

Productivitya, kg m3 h1 3.09 4.46 6.45 12.06

Purity, % 98.51 98.75 98.61 93.21

Yield, % 99.55 99.40 99.47 99.22

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

V.H. Thang, S. Novalin / Bioresource Technology 99 (2008) 43684379 Table 3 Composition of feed solutions Feed 1 2 3 Lactic acid, g L1 260.06 128.78 78.29 Glucose, g L1 76.23 38.74 23.29 Sodium, g L1 71.79 35.55 20.22 Sulfate, g L1 152.45 76.97 44.42

4373

used in further experiments. The composition of feed solution is shown in Table 3. Fig. 5 shows the eect of feed concentration on the separation performance. Like the case of dierent ow-rates, in this case the curves have similar proles and the volume retentions for lactic acid (1.211.35 BV). With the increase

Concentration, g.L-1

from Fig. 4 that that in all experiments, the curves have similar proles and the volume retentions for lactic acid were almost not changed (1.211.33 BV) but the base of the peaks was broader. This will cause a decreasing the resolution between sugar and non-sugar peaks. Data in Table 2 shows that the resolution R decreased from 1.18 to 0.95 with the increasing ow-rate. At low ow-rates of 1.53 and 2.04 cm min1, the dierence between the resolutions was very slightly but the productivity was considerable difference. When the ow-rate increased to 6.12 cm min1 the resolution decreased signicantly. However, in process chromatography the resolution is not always the primordial factor (Stead, 1998). For economical reason, the productivity is rstly considered. As mentioned above, the productivity increased nearly linearly with the increase of ow-rate. However, it should be noted that high ow-rates mean high-pressure drop and signicant dilution. In this work, although the pressure drops were lower than 3 bar, which was still far from the limit for system, the dilution of lactic acid fraction showed no interest in further increasing ow-rate. It can be observed in Fig. 4 that the highest concentration of lactic acid was similar when ow-rate was varying from 1.53 to 3.06 cm min1 but decreased considerably when ow-rate increased to 6.12 cm min1. The data in Table 2 shows that at ow-rates varying from 1.53 to 3.06 cm min1, it seems that the ow-rate had minor eect on the purity. With similar recovery yields, the purity of lactic acid fractions was also similar (around 98.5%). Although it was expected that the purity would decrease with the increase of ow-rate the purity of lactic acid fractions in this case was almost similar. The purity decreased signicantly to 93.21%, only when ow-rate was relatively high (6.12 cm min1). 3.3. Eect of feed concentration on separation Preparative separations are carried out at high concentration under such conditions that the equilibrium isotherms of the feed components in the chromatographic system are non-linear (Felinger and Guiochon, 1996b). However, in this work, the feed concentrations used were still in linear area. The eects of feed concentration on the separation performance of non-lactic and lactic acid were investigated with model solution at three dierent levels using the same feed volume of 10 mL. The obtained results in previous part showed that there was almost no dierence in resolution at ow-rates of 1.53 and 2.04 cm min1, but there was a signicant dierence in productivity. Therefore, the ow-rate of 2.04 cm min1 was

15

12

Concentration, g.L-1

0 0 20 40 60 80 100

Time, min

b
Concentration, g.L-1

15

12

0 0 20 40 60 80 100

Time, min

30

24

18

12

0 0 20 40 60 80 100

Time, min
Lactate Sulfate Glucose Sodium

Fig. 5. Eect of feed concentration on the separation of lactic acid. Conditions: temperature = 60 C, ow-rate = 2.04 cm min1, column: 2.5 100 cm (Dinner H); lactic acid concentration in feed = 78.29 g L1 (a); 128.28 g L1 (b); 260.06 g L1 (c).

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Table 4 Eect of feed concentration on lactic acid separation by chromatography LA concentration in feed, g L1 260.06 128.78 78.29
a

Maximum concentration of LA, g L1 23.00 11.27 6.47

Resolution, R 1.16 1.32 1.45

Productivitya, kg m3 h1 4.46 2.23 1.29

Purity, % 98.75 99.35 99.86

Yield, % 99.40 99.79 99.57

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 lactic acid increased respectively. However, in all cases the ratio between lactic acid concentration in feed and the maximum concentration of lactic acid was similar. It can be seen from Table 4 that the low concentration of feed can improve the purity. With similar recovery yields, the purity increased from 98.75% to 99.86% when the feed concentration decreased from 260.06 g L1 to 78.29 g L1, respectively. Similarly to the purity, the resolution increased signicantly with the decrease of feed concentration. In contrast, the most important parameter for practical operation, namely the productivity, decreased proportionally with the decreasing feed concentration.

3.4. Eect of loaded volumes on separation In production scale, the objective is to obtain a maximum yield with a given degree of separation. The sample should be made as large as possible within the limits set by the required separation. The sample volume could be as large as the separation volume; however, due to nonequilibrium between the mobile phase and longitudinal diffusion in the band, the zones will always be broadened. The sample volume must therefore be smaller that the separation volume. In process chromatography, the feed volume up to 20% of the bed volume can be applied (Stead, 1998). In this work, the loaded volume was investigated with model solutions at three dierent levels: 0.02, 0.04 and 0.10 BV. It can be seen from Fig. 6 that the retention time for lactic acid was shorter when increasing the loaded volume. In the same time, there was a considerable degree of overlap of non-lactic and lactic acid peaks. This overlap zone is very important for the recovery yield and the purity. Table 5 shows details of eects of loaded volume on separation performance. The resolution decreased remarkably, from 1.16 to 0.55 when increasing the loaded volume from 2.2% to 10% of column volume. The maximum concentration of lactic acid increased nearly proportionally to the loaded volume. When the loaded volume was 2.22% of the bed volume, the dierence in concentration between feed and the maximum concentration was the factor of 11.3. This factor was reduced to only 2.4 when the loaded volume reached to 10% of the bed volume. Not only the maximum concentration of lactic acid, but also the productivity was considerably improved by increasing the loaded volume. However, with the similar recovery yields, the purity decreased signicantly 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 chromatography is productivity. However, the recovery yield has to be taken into consideration. In practice, a recovery yield about 90% is usually considered as satisfactory (Felinger and Guiochon, 1996a). Table 6 shows the relation between the recovery yield and other separation parameters. The decrease of recovery yield can improve the purity but not so much. When the recovery yield decreased from 99.50% to 86.77%, the purity increased only from 92.93% to 96.71%. It can be found that a 1% reduction of the recovery yield resulted in about 1% reduction of the productivity. On the other hand, if the recovery yield is lower, the dilution factor as is more improved. The concentration of lactic acid in nal solution (a mixture of collected fractions) increased from 36.20 to 63.14 g L1 when the recovery yield decreased from 99.50% to 86.77%. In practice, the dilution of the product is important because concentrated solutions are required for the commercial products, and therefore, 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 determine the ability of separating lactic acid from a complex mixture by resin XAD1600. The feed used for pulse test was the permeate of nanoltration using membrane MPF-36. The feed volume was 10 mL and the feed composition is presented in Table 7. Fig. 7 presents elution prole of lactic acid separation from silage juice by chromatography. The curve of conductivity shows clearly two separated peaks where the rst peak was much higher than the second one. It proved that the salts were separated from the others. The low conductivity of the second peak proved that almost lactic acid was in undissociated form. This observation agreed with the change of pH. Firstly, the pH of euent decreased together with the increase of inorganic salts because mineral acid, which was used to adjust pH of feed solution, was eluted together with inorganic salts and sugars. After salt fraction, the pH of euent increased slightly, from 1.8 to 3.0, but still lower than pKa of lactic acid. This also showed that lactic 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

V.H. Thang, S. Novalin / Bioresource Technology 99 (2008) 43684379

4375

column. The data shows that lactic acid was satisfactory separated from inorganic salts and sugars, but there was no separation between sugars and inorganic salts and these compounds were eluted together in the rst fraction. The

a
Concentration, g.L-1

30

24

18

resolution R between lactic acid and inorganic salts was 1.63, higher than the highest resolution (R = 1.45) in experiments with model solutions. The high resolution was explained by lower concentration of lactic acid in silage juice when comparing with model solutions. It is known that when the resolution R is 1.5, more than 99% substances are separated. This agreed with the result in adsorption isotherm of salts and sugars on the resin. However, unlike the model solutions, silage juice contains also acetic acid and the purity of lactic acid obtained was aected by this component. Although acetic acid was washed out later than lactic acid, it can be seen that acetic acid was not practically separated from lactic acid. 3.5.2. Preparative separation of lactic acid from concentrated silage juice Unlike analytical chromatography, in which the aim is to separate all individual components of a mixture as completely as possible with subsequent identication of the peaks, in process chromatography the aim of separation often involves detailed economicalchemical optimization calculations. Due to the column dimensions, costs for solvents and packing or pre-packed columns become more and more important with increasing column diameters. For maximizing productivity, process chromatography columns are often overloaded. According to general understanding, industrial scale chromatography is generally carried out under mass and/or volume overloaded conditions in order to increase the product productivity. In volume overloading, the sample concentration is maintained in the linear region of the isotherm and the volume is increased until the productivity is optimized. In mass overloading, the sample concentration is increased beyond the linear adsorption region, resulting in asymmetric band proles. A combination of volume and mass overloading is commonly used to maximize productivity in preparative elution chromatography. However, a concentration overload is only possible, if the solubility of the substances in the feed solution is large enough. In the case of molasses separation, the feed concentration is from 60% to 80% (w/w). However, one important factor, which limits the increase of concentration, is solubility of substances in feed. In this work, silage juice was concentrated until lactate concentration reached about 140 g L1. This concentration was considered the maximum concentration of feed solution since further concentration step might bring diculties, such as high viscosity of solution or crystallization of some substances. The composition of feed mixture is shown in Table 8. Interestingly, acetic acid concentration in concentrated silage juice was similar to that in silage juice. The reason was due to the evaporation of this compound during concentration process of juice. The aim of this work is not optimizing the separation process, therefore the loaded volume was used based on the volume loadability. The volume loadability can be calculated by formula (Scott and Kucera, 1976):

12

0 0 20 40 60 80 100

Time, min

b
Concentration, g.L-1

60

45

30

15

0 0 20 40 60 80 100

Time, min

c
Concentration, g.L-1

120

90

60

30

0 0 20 40 60 80 100

Time, min
Lactate Sulfate Glucose Sodium

Fig. 6. Eect of loaded volume on lactic acid separation from model solutions. Conditions: temperature = 60 C, ow-rate = 2.04 cm min1, column: 2.5 100 cm (Dinner H); Vfeed = 10 mL (a); 20 mL (b); 45 mL (c).

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Table 5 Eect of loaded feed volume on lactic acid separation by chromatography Loaded volume, % of bed volume 2.22 4.44 10.00
a

Maximum concentration of lactic acid, g L1 23.00 48.52 106.38

Resolution, R 1.16 0.90 0.55

Productivitya, kg m3 h1 4.46 8.93 20.29

Purity, % 98.75 97.90 92.93

Yield, % 99.40 99.41 99.50

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

  2 V L V 0 a 1k 0A p 2 k 0A k 0B N

4:2

Table 6 Eect of recovery yield on separation performance Yield, % 99.50 95.36 92.54 86.77 Purity, % 92.93 96.99 96.90 96.71 Productivity 1, kg m3 h1 20.29 19.45 18.77 17.69 Lactic acid concentration, g L1 36.20 40.82 51.80 63.14 Dilution factor 7.2 6.4 5.0 4.1

where VL is the maximum load volume; V0 is the dead volume; a is relative retention; k 0A and k 0B are capacity factors and N is the theoretical plate number. As can be seen from the formula (4.2), the volume loadability linearly depends on the dead volume of the column used, and otherwise it only depends on the k 0 values of the components to be separated and on the separation eciency of the column. From obtained data in pulse tests, the volume loadability was calculated as about 40 mL for above column. Fig. 8 presents elution prole of lactic acid separation from 4-fold concentrated silage juice by chromatography. It was found that the behavior of conductivity was not

Table 7 Composition of silage juice used as feed for pulse test Substance Lactic acid Acetic acid Amino acids Sugars (glucose, fructose, sucrose, arabinose, xylose, . . .) Inorganic salts (K+, Na+, Ca2+, Mg2+, NH , Cl, 4 NO ; SO2 ) 3 4 Concentration, g L1 35 2.0 22.8 26.85 62.78

Table 8 Composition of concentrated silage juice as feed for chromatographic separation Substance Lactic acid Acetic acid Amino acids Sugars (glucose, fructose, sucrose, arabinose, xylose, . . .) Inorganic salts (K+, Na+, Ca2+, Mg2+, NH , Cl, 4 NO ; SO2 ) 3 4 Concentration, g L1 140.0 2.0 90.0 105.2 249.1

10

16 14

120

10

pH Conductivity, mS.cm-1

Concentration, g.L-1

12 6 10 8 4 6 4 2 2 0 0.0 0.5 1.0 1.5 2.0 0 2.5

100

Conductivity, mS.cm-1 Concentration, g.L-1

80 6 60 4 40 20 0 0.0 0.5 1.0 1.5 2.0 2.5 2

Bed volume
Inorganic salts Acetic acid Sugars pH Lactic acid Conductivity

Bed volume
Sugars Inorganic salts Lactic acid Conductivity Acetic acid pH

Fig. 7. Elution prole of lactic acid separation from silage juice by chromatography. Conditions: Vfeed = 10 ml, ow-rate = 2.04 cm min1, T = 60 C, column: 2.5 100 cm (Dinner H).

Fig. 8. Separation of lactic acid from concentrated silage juice by preparative chromatography. Conditions: Vfeed = 40 ml, ow-rate = 2.04 cm min1, T = 60 C, column: 2.5 100 cm (Dinner H).

pH

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similar to that in pulse test experiment: only one peak was observed instead of two. The lactic acid peak could not be seen based on the curve of conductivity. These results proved that the higher volume is loaded, the bigger overlap area will be resulted. Therefore, inorganic salts in the overlap area caused the conductivity higher than that caused by undissociated lactic acid. Like experiments with model solutions, lactic acid was completely eluted after 1.7 BV of eluent. Although the resolution between lactic acid and inorganic salts slightly decreased in comparison with the result obtained in pulse test, the separation between these compounds was still very good. However, the problem in separating lactic acid from acetic was still remained. It can be seen in Fig. 8 that acetic acid was eluted nearly together with lactic acid. Unfortunately, due to the very complex composition, silage juice contains not only acetic acid but also other organic acids with smaller amounts. These organic acids caused the same problem in separating lactic acid from other organic acids. Besides acetic acid, amino acids also occurred in lactic acid fraction as impurities. Fig. 9 shows that amino acids occurred in all collected fractions. In lactic acid fraction, the main remaining amino acids were valine, leucine, isoleucine and lysine. After the separation, two main fractions were obtained: lactic and non-lactic fraction. Lactic acid fraction has concentration of 17.94 g L1 with the recovery yield of 97% and purity of 94%. More than 99% of inorganic salts and sugars were separated from lactic acid. The composition of lactic acid fraction obtained after separation process was analyzed in HPLC. The result is presented in Fig. 10, where besides lactic acid and acetic acid other organic acids such as citric and succinic acids are existed. Besides the purity problem, another disadvantage was the dilution of product. In this investigation, the dilution factor was about 7.9. However, it was quite dicult to improve the dilution factor. It seems that the loaded volume was close to the limitation for column separation. Moreover, unlike the case of model solutions, the concentrated silage juice has a limitation in maximum lactic acid concentration.
20

3.5.3. Proposed processes for separation of lactic acid from silage juice From the obtained results, a possible process for separating lactic acid from grass silage juice is proposed as Fig. 11. Silage juice has to be concentrated before being further processed. The solubility of concentrated solution should be taken into account. A concentration factor of four of vefolds is suggested. This concentration step can also reduce a great part of acetic acid, thus minimize its amount in the lactic acid product. The acidication step is done by using mineral acids, but sulfuric acid is preferred. When concentrated sulfuric acid is used for acidifying the juice, the feed for chromatography will be less diluted. Other advantages of using sulfuric acid are that it is cheaper and less corrosive than other mineral acids. The acidied juice is then applied to a chromatography column containing neutral polymeric resin like XAD1600. Lactic acid was then eluted from the column by using water that is condensed from evaporation steps. However, if lactic acid is produced like this process, a part of amino acids will be still remained as impurities. For the resin, it was observed that colored components were adsorbed into the resin. Although in this work, this phenomenon seems not aect the performance of lactic acid separation, the more detailed investigation about the eect of a full-color-adsorbed column on the lactic acid separation is needed. The problem of amino acid as impurity could be minimized by using chromatography in combination with electrodialysis. Thang et al. (2004) reported that about 90% of amino acids were separated from lactate fraction by electrodialysis. Thus, an alternative process, which combines chromatography and electrodialysis could be proposed (Fig. 11). Firstly, amino acids will be separated by electrodialysis because at neutral pH, most of amino acids are in neutral forms and therefore they will be remained in the diluate of electrodialysis. The concentrate of electrodialysis containing mainly charged components such as lactic acid and inorganic salts will further be processed like proposed scheme in Fig. 11.

Group 1 16

Group 1: Aspartate, Glutamate,

Group 2

Concentration, g.L-1

Asparagine, Glutamine, Histidine, Glycine, Threonine, Arginine, Alanine, Gaba. Group 2: Valine, Methionine. Group 3: Tyrosine, Leucine, Isoleucine, Lysine.

12

Group 3

0 0.0

0.5

1.0

1.5

2.0

2.5

Bed volume

Fig. 9. Euent proles of amino acids for XAD1600 resin. Conditions: Vfeed = 40 ml, ow-rate = 2.04 cm min1, temperature = 60 C, column: 2.5 100 cm (Dinner H).

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5 - Lactate

V.H. Thang, S. Novalin / Bioresource Technology 99 (2008) 43684379

Fig. 10. HPLC chromatogram of the lactic acid separated from grass silage juice.

ganic salts and sugars were separated from lactic acid, organic acids in general and acetic acid in particular caused a purity problem. Besides the organic acids, some amino acids were remained in the products as impurities. For the production of lactic acid from raw materials having less purity, the above-mentioned method should further be investigated more in detail. However, the results from this work can show a good hint for a single-step separation of lactic acid produced from quite pure raw materials, such as glucose syrup or starchy hydrolysates. In these cases, the fermentation broths will contain much less free amino acids and organic acids. Finally, due to the dilution of product, more energy consumption will be needed for concentration step to obtain commercial product. Acknowledgements This research has been carried out in connection with the programme Fabrik der Zukunft of the Austrian Federal Ministry of Trac, Innovation and Technology (Pro ject No.806097). The authors would like to thank OAD (Osterreichischer Austauschsdienst/Austrian Academic Exchange Service) for granting V.H. Thang a doctoral scholarship. References
Bailly, M., 2002. Production of organic acids by bipolar electrodialysis: realizations and perspectives. Desalination 144, 157162. Cao, X., Yun, H.S., Koo, Y.M., 2002. Recovery of L-(+)-lactic acid by anion exchange resin Amberlite IRA-400. Biochemical Engineering Journal 11, 189196. Choi, J.I., Hong, W.H., 1999. Recovery of lactic acid by batch distillation with chemical reactions using ion exchange resin. Journal of Chemical Engineering of Japan 32, 184189. Danner, H., Madzingaidzo, L., Holzer, M., Mayrhuber, L., Braun, R., 2000. Extraction and purication of lactic acid from silages. Bioresource Technology 75, 181187. Datta, R., Tsai, S.P., Bonsignore, P., Moon, S.H., Frank, J.R., 1995. Technological and economic potential of poly (lactic acid) and lactic acid derivatives. FEMS Microbiology Reviews 16, 221231. Dechow, F.J., 1991. Industrial ion exchange chromatography. In: Dorfner, K. (Ed.), Ion Exchangers. Walter de Gruyter, New York, pp. 10291059. Evangelista, R.L., Nikolov, Z.L., 1996. Recovery and purication of lactic acid from fermentation broth by adsorption. Applied Biochemistry and Biotechnology. 57/58, 471480. Evangelista, R.L., Mangold, A.J., Nikolov, Z.L., 1994. Recovery of lactic acid by sorption: resin evaluation. Applied Biochemistry and Biotechnology 45/46, 131144. Felinger, A., Guiochon, G., 1992a. Optimization of the experimental conditions and the column design parameters in overloaded elution chromatography. Journal of Chromatography A 591, 3145. Felinger, A., Guiochon, G., 1992b. Optimization of the experimental conditions and the column design parameters in displacement chromatography. Journal of Chromatography A 609, 3547. Felinger, A., Guiochon, G., 1996a. Optimizing preparative separations at high recovery yield. Journal of Chromatography A 752, 3140. Felinger, A., Guiochon, G., 1996b. Optimizing experimental conditions in overloaded gradient elution chromatography. Biotechnology Progress 12, 638644. Forschungsforum, 1/2004. Green Biorenery: Ein Innovatives Technologiekonzept zur Schaung einer nachhaltigen Rohstobasis im

2 - Glucose 3 - Fructose

4 - Succinate

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

4. Conclusions This work has shown that the chromatographic separation using neutral adsorbent resins like Amberlite XAD1600 could simply solve the desalting problem in recovery of lactic acid from grass silage juice. More than 99% of inorganic salts as well as sugars were removed with recovery yields over 99.4%. However, due to the complex composition of the grass silage juices, the purity of lactic acid decreased to 94% at the recovery yield of 97%. Although about 99% of inor-

1 - Citrate

6 - Acetate

V.H. Thang, S. Novalin / Bioresource Technology 99 (2008) 43684379 Rahmen von, Fabrik de Zukunft. Forschungforum 1/2004. Projektfabrik, Austrian Federal Ministry for Transport, Innovation and Technology. Guiochon, G., 2002. Preparative liquid chromatography. Journal of Chromatography A 965, 129161. Heriban, V., Skara, J., Sturdik, E., Ilavsky, J., 1993. Isolation of free lactic acid using electrodialysis. Biotechnology Techniques 7, 63 68. Kromus, S., Wachter, B., Koschuh, W., Mandl, M., Krotscheck, C., Narodoslawsky, M., 2004. The Green Biorenery Austria Development of an integrated system for Green Biomass utilization. Chemical and Biochemical Engineering Quarterly 18, 712. Kulprathipanja, S., 1988. Separation of citric acid from fermentation broth with a neutral polymeric adsorbent. US Patent 4,720,579. Madzingaidzo, L., Danner, H., Braun, R., 2002. Process development and optimisation of lactic acid purication using electrodialysis. Journal of Biotechnology 96, 223239. Matsumoto, M., Takahashi, T., Fukushima, K., 2003. Synergistic extraction of lactic acid with alkylamine and tri-n-butylphosphate: eects of amines, diluents and temperature. Separation and Purication Technology 33, 8993. Ponnampalam, E., 2001. Purication of organic acids using anion exchange chromatography. US Patent, US6284904B1.

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Rohm and Hass Company, 2002. Product data sheet of Amberlite XAD1600, industrial grade polymeric adsorbent. Scott, R.P.W., Kucera, P., 1976. Some aspects of preparative-scale liquid chromatography. Journal of Chromatography A 119, 467482. Stead, P., 1998. Isolation by preparative chromatography. In: Cannell, Richard J.P. (Ed.), Natural Products Isolation. Humana Press Inc., Totowa, NJ, USA, pp. 165208. Thang, V.H., Koschuh, W., Kulbe, K.D., Kromus, S., Krotscheck, C., Novalin, S., 2004. Desalination of high salt content mixture by twostage electrodialysis as the rst step of separating valuable substances from grass silage. Desalination 162, 343353. Thang, V.H., Koschuh, W., Kulbe, K.D., Novalin, S., 2005. Detailed investigation of an electrodialytic process during the separation of lactic acid from a complex mixture. Journal of Membrane Science 249, 173182. Tong, W.Y., Fu, X.Y., Lee, S.M., Yu, J., Liu, J.W., Wei, D.Z., Koo, Y.M., 2004. Purication of L-(+)-lactic acid from fermentation broth with paper sludge as a cellulosic feedstock using weak anion exchanger Amberlite IRA-92. Biochemical Engineering Journal 18, 8996. Yankov, D., Molinier, J., Albet, J., Malmary, G., Kyuchoukov, G., 2004. Lactic acid extraction from aqueous solutions with tri-n-octylamine dissolved in decanol and dodecane. Biochemical Engineering Journal 21, 6371.