Bioresource Technology 193 (2015) 250–255

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Production of technical-grade sodium citrate from glycerol-containing biodiesel waste by Yarrowia lipolytica Svetlana V. Kamzolova, Natalia G. Vinokurova, Julia N. Lunina, Nina F. Zelenkova, Igor G. Morgunov ⇑ G.K. Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Pushchino 142290, Russia

h i g h l i g h t s  Sodium citrate is a substitute for ecologically safe phosphate-free detergents.  The production of sodium citrate from glycerol-containing biodiesel waste by yeast.  Fermentations were carried out by methods of batch- and repeated batch cultivation.  Repeated batch fermentation provides a high biosynthetic activity for more than 20 days.  The product was isolated from the culture broth by simple procedure.

a r t i c l e

i n f o

Article history: Received 29 April 2015 Received in revised form 19 June 2015 Accepted 20 June 2015 Available online 25 June 2015 Keywords: Yarrowia lipolytica yeast Citric acid production Sodium citrate Phosphate-free detergents Biodiesel-derived waste glycerol

a b s t r a c t The production of technical-grade sodium citrate from the glycerol-containing biodiesel waste by Yarrowia lipolytica was studied. Batch experiments showed that citrate was actively produced within 144 h, then citrate formation decreased presumably due to inhibition of enzymes involved in this process. In contrast, when the method of repeated batch cultivation was used, the formation of citrate continued for more than 500 h. In this case, the final concentration of citrate in the culture liquid reached 79–82 g/L. Trisodium citrate was isolated from the culture liquid filtrate by the addition of a small amount of NaOH, so that the pH of the filtrate increased to 7–8. This simple and economic isolation procedure gave the yield of crude preparation containing trisodium citrate 5.5-hydrate up to 82–86%. Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction Sodium triphosphate is still the main component of most synthetic detergents. As a result, high amounts of phosphorus appeared in water bodies with sewage and promoted intense growth of blue-green algae (cyanobacteria). These organisms are capable of photosynthesis and nitrogen fixation, due to which their growth does not depend on the presence of carbon sources and bound nitrogen in the medium. This fact gives great advantage to cyanobacteria over other bacteria. The fast growth of cyanobacteria is harmful to the environment due to their ability to synthesize various secondary metabolites (cyclopeptides, alkaloids, etc.) which are deadly toxic to fishes, birds, animals and humans (Catherine et al., 2013; Zanchett and Oliveira-Filho, 2013; Boopathi and Ki, 2014). The high risk of phosphates forced many ⇑ Corresponding author at: G.K. Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Prospect Nauki 5, Pushchino, Moscow Region 142290, Russia. Tel.: +7 4967 318660. E-mail address: [email protected] (I.G. Morgunov). http://dx.doi.org/10.1016/j.biortech.2015.06.092 0960-8524/Ó 2015 Elsevier Ltd. All rights reserved.

countries to restrict their content in waste waters, whereas some of them (for example, Austria, Germany, Italy, Netherlands, Norway, USA, Switzerland, and Japan) totally prohibited the production of phosphate-containing detergents. The substitution of polyphosphates with citrates in the production of detergents seems to be a promising approach, since citrates are not corrosive and can be easily degraded to carbon dioxide and water in the waste water treatment plants. Sodium citrate is usually produced from citric acid (CA) by using such stages as acid dissolution, salt formation, and salt isolation. CA is mainly produced by the mycelial fungus Aspergillus niger from molasses, the sugar refinery waste. The sugar content of molasses does not exceed 50%; the rest contains various ballast substances which are utilized by the producer. Another disadvantage of this production method is the necessity of molasses treatment with ferrocyanides to remove the excess of trace elements which are harmful to A. niger. As a result, the production of citric acid by this method is complicated and ecologically dangerous. The aforementioned poses the problem of seeking for new renewable carbon sources and organisms suitable for the

S.V. Kamzolova et al. / Bioresource Technology 193 (2015) 250–255

production of citric acid. One of the promising organisms in this respect is the yeast Yarrowia lipolytica. As for the inexpensive carbon sources, recent studies showed the promise of the biodiesel-derived waste glycerol for microbiological production of valuable metabolites. On the basis of this waste glycerol, researchers developed the method of production of 1,3-propanediol (Papanikolaou et al., 2008), ethanol (Liu et al., 2012), polyhydroxybutyrate (Mothes et al., 2007), biomass enriched in essential amino acids (Juszczyk et al., 2013), lipids (Dedyukhina et al., 2012; Munch et al., 2015), polyunsaturated fatty acids (Dedyukhina et al., 2014), erythritol (Miron´czuk et al., 2014), carbonic acids, in particular, propionic acid (Tufvesson et al., 2013), pyruvic acid (Morgunov et al., 2004), a-ketoglutaric acid (Yin et al., 2012; Otto et al., 2012), and citric acid (Rymowicz et al., 2006, 2010; Förster et al., 2007; Holz et al., 2009; Kamzolova et al., 2011; Morgunov et al., 2013). The aim of this study was to develop microbiological method suitable for the production of technical-grade sodium citrate from the glycerol-containing biodiesel waste with the use of the yeast Y. lipolytica. Such technical sodium citrate can be applied for the substitution of ecologically harmful sodium triphosphate, a component of synthetic detergents.

2. Methods Experiments were carried out with the specially selected yeast strain Y. lipolytica VKM Y-2373, which is able to produce citric acid from the glycerol-containing waste of biodiesel manufacture (Kamzolova et al., 2011; Morgunov et al., 2013). Batch cultivation conditions, methods of biomass determination and analysis of CA and ICA (isocitric acid), as well as the calculation of the quantitative parameters of fermentation, were described earlier (Morgunov et al., 2013). We have previously carried out experiments on optimization of the fermentation process of citric acid production from the glycerol-containing biodiesel waste by Y. lipolytica yeast (Morgunov et al., 2013). It was shown that the cultivation condition, ensuring the maximum rate of citric acid production were as follows: limitation of cell growth by nitrogen (at initial C/N ratio equal to 7.8:1), the temperature of 28 °C, pH 5; oxygen supply of 50% of saturation. The cultivation medium was supplemented with biodiesel waste containing 80% glycerol as the source of carbon and energy. The regime of biodiesel waste supplement included the initial addition of waste (by 20 g/L) and then by 20 g per an entire fermenter volume. The substrate feeding was carried out synchronously with the sharp decrease in a respiratory activity of cells. A decrease in a respiratory activity of cells was accompanied by an increase in the level of pO2 by 5–10% from the constant level during the process. Such increase in pO2 indicated the complete consumption of biodiesel waste by cells. Repeated batch cultivation was carried out as follows: a part of the culture liquid containing yeast cells and excreted metabolites was periodically removed from a fermentor and replaced by the same volume of fresh nutrient medium. The fermentor always contained 5 L of fermentation liquid. The percentage of medium input was varied from 20% to 50% with a step of 10%. Yeast growth was followed by measuring the biomass dry weight: 1–3 mL of the culture broth was filtered through a membrane filter and the yeast cells were washed with n-hexane and dried in a vacuum desiccator at 110 °C to a constant weight. To analyze organic acids, the culture broth was centrifuged (8000g, 20 °C, 3 min), 1 mL of the supernatant was diluted with an equal volume of 8% HClO4 and concentration of organic acids was measured on an HPLC-chromatograph (LKB, Sweden) on an Inertsil ODS-3 reversed-phase column (250  4 mm, Elsiko, Russia) at 210 nm; 20 mM phosphoric acid was used as a mobile

251

phase with the flow rate of 1.0 mL min 1; the column temperature was maintained at 35 °C. Quantitative determination was carried out using calibration curves constructed with the application of CA, threo-Ds-isocitric acid (ICA) and other acids (Sigma–Aldrich, St. Louis, MO, USA) as standards. Glycerol was analyzed enzymatically using biochemical kit (Roche Diagnostics GmbH, Germany). The biomass yield (Yx/s), the specific growth rate (l), the mass yield coefficient of CA production (Yp), the volumetric productivity (Qp) were calculated as described earlier (Morgunov et al., 2013). The activity of enzymes in the cell-free homogenate was determined as described earlier (Kamzolova et al., 2011; Morgunov et al., 2013). To isolate sodium citrate, 450 mL of the culture liquid filtrate, containing 36.3 g CA and 8.6 g ICA, was supplemented with 2 g of dry activated charcoal, which was preliminarily washed with hydrochloric acid to remove iron ions and then washed with water and dried. The mixture was heated in a water bath to 70 °C and incubated for 30 min. Then the mixture was alkalified to pH  7.6 by the addition of 40% sodium hydroxide. The used charcoal was harvested by filtration and washed with water. The colorless filtrate combined with washing waters was evaporated at a temperature below 50 °C using a rotary evaporator, until the volume decreased to 80–85 mL and the concentrated liquid looked like syrup. Then the concentrated liquid was cooled to 10–12 °C under agitation, which initiated the precipitation of the sodium salts of CA and ICA. The agitation was continued for 3 h to increase the yield of the salts. The salt crystals were collected by filtration and dehydrated by compression. The filtrate was further concentrated to 30–35 mL by evaporation and used for the second stage of preparation of CA and ICA salts in the manner described above. Both portions of salts were combined and dried at 40–50 °C under periodical stirring. The resultant crystalline material was crushed to powder in a mortar. The weight of the salts obtained was 64.8 g.

3. Results and discussion 3.1. Production of CA by batch cultivation Fig. 1a shows the time courses of Y. lipolytica VKM Y-2373 growth, nitrogen consumption, and the accumulation of CA and ICA. The cell growth was very poor for the first three hours and became linear during the next 12 h (in the course of exponential phase) because the cultivation medium contained sufficient supply of nutrients. The cultivation parameters were close to ideal growth conditions, the maximal specific growth rate (lmax) was equal to 0.23 h 1. After 15 h of cultivation, the culture passed to the phase of growth retardation, the specific growth rate gradually decreased to 0.07 h 1; after 36 h of cultivation, the culture growth was ceased. Acid formation was absent in the exponential growth phase, started in the phase of growth retardation, and actively continued in the stationary growth phase. By the end of the experiment (192 h of cultivation), Y. lipolytica VKM Y-2373 accumulated 92 g/L CA and 15.3 g/L ICA (a ratio CA:ICA was 6:1). Calculations showed that the culture productivity and the product yield were 0.766 g/L h and 63%, respectively. According to literature data, glycerol-grown yeast Y. lipolytica NRRL YB-423 produced 21.6 g/L of CA with the product yield of 54% (Levinson et al., 2007); strain Y. lipolytica ACA-DC 50109 synthesized 62.5 g/L of CA with the product yield of 56% (Papanikolaou et al., 2008). Mutant Y. lipolytica Wratislavia AWG7 grown on biodiesel-derived glycerol produced up to 131.5 g/L of CA, and a CA:ICA ratio was 29:1 (Rywin´ska et al., 2009). As is seen from Fig. 1b, the culture productivity reached the maximum in the phase of growth retardation and began to

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(a)

biomass (X)

CA

ICA

formation during the batch cultivation of Y. lipolytica coincides with a decrease in the activities of enzymes involved in the oxidation of glycerol and synthesis of citric acid. This can be due to the inhibitory action of CA or to the aging of cells suffering from the deficiency of nitrogen in the medium.

N

900

80 60

600

40

N (mg/L)

Biomass, CA, ICA (g/L)

100

300 20 0 0

24

48

72

96

120

144

168

0 192

Time (h)

(b)

µ

log X

qp

0,2

0,7

0,15

0,2

0,1

-1

LogX

1,2

µ (h ), qp (g CA/g cells h)

0,25

1,7

-0,3

0

24

48

72

96

120

144

168

1920,05 0

-0,8 Time (h)

Fig. 1. Growth of Y. lipolytica, nitrogen consumption, the accumulation of CA and ICA (a), and the calculated parameters of fermentation (,).

decrease after 120–140 h of cultivation, when the content of CA was 70–80 g/L. This fact indicates that the optimal cultivation time is 5–6 days. The morphological studies of the cell population of Y. lipolytica showed that during the period of active acid formation (36–120 h of cultivation), the yeast cells were round, oval, or elongated. The shape of some cells was irregular, the number of petite cells comprised about 15%. The cellular protoplasm was dense and granular. Each cell contained 1–2 lipid granules. Vacuoles were well developed, especially in the cells of irregular shape. The number of cells with plasmolysis did not exceed 6–7%. When the activity of acid formation decreased (after 120 h of cultivation), the number of petite cells gradually increased and reached 30–35% by the end of cultivation (192 h of growth). In this case, the number of dead plasmolyzed cells reached 20–25%. To find the reason for drop in the production of CA in the course of batch fermentation, we measured the activities of the key enzymes of glycerol and CA metabolism in the cell homogenate of Y. lipolytica VKM Y-2373. The cell culture was sampled at the time of intense acid formation (36 h of cultivation) and the low acid formation (120 and 192 h of cultivation). As seen from the data shown in Table 1, the activity of glycerol kinase, the first enzyme of glycerol metabolism, was decreased in the process of fermentation by 2.5 times. The activities of citrate synthase, aconitate hydratase, NAD- and NADP-dependent isocitrate dehydrogenases were also decreased, a decrease in activities of the last two enzymes was especially pronounced (by 9 and 6 times, respectively). This fact can be explained by lowering the function of the tricarboxylic acid (TCA) cycle. As a whole, the drop in the acid

3.2. Investigation of the inhibitory effect of CA The experiments were carried out as follows: the yeast culture was taken from a fermentor in the phase of active acid formation (36 h of cultivation). The cells were harvested by centrifugation, washed twice with 0.9% NaCl, and suspended in 50 mM phosphate buffer (pH 7,0). The cell suspension was placed in 750-mL Erlenmeyer flasks with 50 mL of the Reader medium containing (g/L): glycerol, 10.0; MgSO4
7H2O, 0.7; NaCl, 0.5; Ca(NO3)2, 0.4; KH2PO4, 1.0; K2HPO4, 0.1; and various amounts of CA (from now on, this deliberately added CA is named exogenous CA to avoid confusion with the CA formed endogenously by the yeast cells). Taking into account the fact that the final density of cells in the flasks was 3 g/L, as compared to 20 g/L in the fermentor, the concentration of exogenously added CA in the flasks equal to 12 g/L corresponded to 80 g/L observed during fermentation in a fermentor. The flasks were incubated under shaking at 29 °C for 24 h. The results are shown in Fig. 2. As seen from this figure, concentration of exogenous CA in a range of 1–12 g/L, does not inhibit but even stimulates the biosynthesis of endogenous CA by the yeast cells. With the further increase in the concentration of added CA (up to 40 g/L), the biosynthesis of CA decreased twofold. In this case, the microscopic examination of yeast cells and the measurement of extracellular protein showed that the lysis of cells was insignificant. Thus, CA at concentrations which are accumulated in a fermentor shows no effect on acid formation. It can be suggested that it is necessary to regenerate the cell population of Y. lipolytica in order to maintain its acid productivity. A possible approach is described below. 3.3. Repeated batch fermentation One of the promising approaches to overcome the aforementioned difficulties of batch fermentation is a repeated batch fermentation (Anastassiadis and Rehm, 2006; Rywin´ska and Rymowicz, 2010; Rymowicz et al., 2010). The repeated batch fermentation is a process when part of the culture liquid containing yeast cells and excreted metabolites is periodically removed from a fermentor and the same volume of fresh nutrient medium is fed into a fermentor. In our experiments, we varied the volume of refreshed medium and the number of cyclic refreshments (feeding). The experimental variants included: (1) 20% feeding, 2 cycles; (2) 30% feeding, 2 cycles; (3) 50% feeding, 1 cycle; (4) 40% feeding, 2 cycles. The duration of fermentation was 521 h. The duration of one cycle was varied from 41 to 102 h. The feedings were made when the concentration of CA in the medium reached about 80 g/L. As seen from Fig. 3, even after 521 h of cultivation, the concentration of CA in the medium was as high as 80.7 g/L. Table 2 shows the mean values of biomass, CA, ICA, as well as the calculated parameters characterizing the production of CA, namely, volume productivity (CCA), culture productivity (qp), and the yield of CA relative the glycerol consumed. As seen from Table 2, in all the regimes studied, the culture liquid contained 7 –82.1 g/L CA at the moments of culture feeding and the amount of ICA did not exceed 20% of the sum CA + ICA. The volume productivity and the yield of CA were maximal (1.001 g/L h and 70%, respectively) when the culture was refreshed every 48 h to a degree of 40%. These production parameters were higher than

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S.V. Kamzolova et al. / Bioresource Technology 193 (2015) 250–255 Table 1 Activities of enzymes of the tricarboxylic acid cycle and the glyoxylate cycle during Y. lipolytica VKM Y-2373 growth and CA production on biodiesel-derived glycerol. Time (h)

GK

TCA cycle

36 120 192

0.112 ± 0.020 0.107 ± 0.015 0.044 ± 0.005

Glyoxylate cycle

CS

AH

NAD-ICDH

NADP-ICDH

IL

MS

1.74 ± 0.20 1.60 ± 0.16 0.60 ± 0.10

0.12 ± 0.01 0.09 ± 0.01 0.06 ± 0.01

0.090 ± 0.010 0.030 ± 0.003 0.010 ± 0.001

0.080 ± 0.010 0.090 ± 0.010 0.010 ± 0.003

0.089 ± 0.010 0.090 ± 0.010 0.073 ± 0.008

0.035 ± 0.004 0.030 ± 0.030 0.020 ± 0.002

Enzyme activities are expressed in U/mg protein. Abbreviations: GK, glycerol kinase; CS, citrate synthase; AH, aconitate hydratase; NAD-ICDH, NAD-dependent isocitrate dehydrogenase; NADP-ICDH, NADP-dependent isocitrate dehydrogenase; IL, isocitrate lyase; MS, malate synthase.

Endogenous CA (g/L)

5 4 3 2 1 0 0

5

10

15

20

25

30

35

40

45

Exogenous CA (g/L) Fig. 2. The effect of added CA on the biosynthesis of endogenous CA by Y. lipolytica.

the respective parameters of batch fermentation by 30.6% and 11.1%, respectively. The rate of CA biosynthesis (0.034 g/L h) in the regime of repeated batch fermentation corresponded to the maximal value of this parameter in the regime of simple batch fermentation (within 72–132 h of batch cultivation). An increase in the feeding value from 40% to 50% reduced the efficiency of the process; namely, the time of the CA concentration reversion to the basal value 80 g/L increased from 48 to 102 h; the volume productivity CCA and the culture productivity qp reduced by 2.5 and 1.7 times, respectively; the yield of CA was 1.1 times lower. Similarly, a decrease in the feeding value from 40% to 20% reduced the volume productivity CCA, the culture productivity qp, and the yield of CA by 3.1, 2.1, and 7%, respectively. The method of repeated batch fermentation has been already described in the literature. Arzumanov et al. (2000a,b) developed

biomass

CA

the process which maintained the acid formation ability of the culture grown on ethanol for more than 700 h. Anastassiadis and Rehm (2006) successively carried out 20 repeated batch fermentations of Candida oleophila in the medium with glucose as the source of carbon and energy. The authors did not meet any difficulties with cultivation or the maintenance of culture in the physiologically active state. Rywin´ska and Rymowicz (2010) described the repeated batch cultivation of Y. lipolytica Wratislavia 1.31 and Wratislavia AWG7 for 1350 and 1680 h, respectively, in the medium with glycerol-containing waste. The maximal concentrations of CA in the medium were reached when the feeding value was 20%; namely, these concentration were equal to 197 and 176 g/L for Y. lipolytica Wratislavia AWG7 and Y. lipolytica Wratislavia 1.31, respectively. It should be noted, however, that both strains produced notable amounts of byproduct erythritol (15.5 and 17.7 g/L, respectively). Makri et al. (2010) showed the formation of CA and lipids by Y. lipolytica cultivated on glycerol-containing waste in the repeated batch mode. Thus, experiments described in this article and those available in the literature indicate that the repeated batch cultivation is able to maintain the active synthesis of CA and probably other products at high levels for a long time. 3.4. Separation and purification of sodium citrate from the culture liquid The filtrate of the culture liquid with pH 5–6 mainly contained di- and trisodium salts of citric and isocitric acids in a proportion of 4.2:1 (relative to citric and isocitric residues) and some amount of inorganic salts. Trisodium CA and ICA salts were isolated directly from the culture liquid filtrate by the addition of a small amount of NaOH, so that the pH of the filtrate increased to 7–8. This simple

ICA

Biomass, CA, ICA (g/L)

80

60

40

20

0 0

48

96

144

192

240

288

336

384

432

480

Time (h) Fig. 3. Durable cultivation of Y. lipolytica in the regime of repeated batch cultivation.

528

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S.V. Kamzolova et al. / Bioresource Technology 193 (2015) 250–255

Table 2 Biosynthesis of CA by Y. lipolytica VKM Y-2373 cultivated in the repeated batch mode. Feeding (%)

Cycle duration (h)

Biomass (g/L)

CA (g/L)

ICA (g/L)

CCA (g/L h)

qp (g/g h)

YCA (%)

20 30 50 40

48 41 102 48

20.5 ± 1.5 20.4 ± 1.3 19.7 ± 1.9 19.0 ± 1.7

80.3 ± 2.2 82.1 ± 2.2 79.0 ± 1.5 80.7 ± 2.2

19.3 ± 2.0 19.4 ± 1.9 18.4 ± 2.0 19.1 ± 1.9

0.327 0.588 0.401 1.001

0.016 0.029 0.020 0.034

65 68 63 70

Table 3 Comparison of sodium citrate production by the yeast Y. lipolytica from glycerolcontaining biodiesel waste and by the fungus A. niger from molasses. Y. lipolytica

A. niger

1. 2. 3. 4. 5. 6. 7. 8.

1. Treatment of molasses 2. Fermentation 3. Biomass collection 4. Treatment with CaCO3 5. Separation and washing of Ca-citrate 6. Treatment with H2SO4 7. Removal of calcium sulfate 8. Discoloring 9. Alkalification 10. Evaporation 11. Crystallization 12. Filtration 13. Drying

Fermentation Biomass collection Discoloring Alkalification Evaporation Crystallization Filtration Drying

properties, in particular, trimethyl citrate is in a solid state at melting point of 75 °C, while trimethyl isocitrate proved to be a liquid under these conditions. This finding made it possible to separate both acid esters by simple filtration. 4. Conclusion The advantages and disadvantages of processes of sodium citrate production by A. niger and Y. lipolytica are compated in Table 3. The former process is ecologically harmful and involves 13 stages, including the treatment of molasses with ferrocyanides. In contrast, the production of sodium citrate with the use of Y. lipolytica is ecologically safe and includes only 8 stages. Sodium citrate is isolated directly from the culture liquid filtrate without the use of concentrated mineral acids and alkalies. Conflict of interest

and economic isolation procedure gave a crude preparation containing trisodium citrate 5.5-hydrate and trisodium isocitrate in a proportion of about 6:1. The problem which we meet was in that the amount of crystal water in the prepared trisodium ICA was unknown. In attempts to solve this problem, we heated the salt mixture at 110 °C to a constant weight; however, dehydration at this temperature was incomplete. The attempt to dehydrate the preparation at 240 °C (at this temperature, trisodium CA is totally dehydrated) was also unsuccessful, because trisodium ICA at 240 °C was decomposed, which was evident from the fact that the salt preparation become dark. The analysis of the preparation by HPLC showed that it contained 82–86% of trisodium citrate 5.5-hydrate (this amount corresponded to the yield of CA equal to 77–83%). The residue (14–18%) was probably represented by trisodium ICA, because HPLC did not show the presence of other organic compounds in the salt preparation. Since sodium isocitrate is similar in its structure to sodium citrate, it could be assumed that their properties were also similar. Therefore, the presence of the mixture of these salts in the preparation is quite permissible and no expenditure for the salt separation is required. The presence of inorganic impurities in the preparation was negligible and did not prevent the use of technical trisodium citrate for production of phosphate-free detergents. It should be noted that production of sodium citrate with the use of Y. lipolytica cultivated on ethanol (Arzumanov et al., 2000a,b; Aghajanyan, 2005; Aghajanyan et al., 2007) meets serious technical difficulties, especially at an industrial scale; although the combination of electrodialysis and the sorption methods allowed the authors (Aghajanyan, 2005; Aghajanyan et al., 2007) to prepare 99.5% crystalline sodium citrate dihydrate with the yield of 87%. Heretsch et al. (2008) and Aurich et al. (2012) noted that citricand isocitric acids are constitutional isomers and have the same functional groups. This means that physicochemical properties of these acids, such as polarity, acidity, and solubility are similar that creates the problems of separation of these acids. However, the methyl esters of these acids are characterized by different

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