Optimization of Process Conditions for Lactic Acid Recovery Using ...

Optimization of Process Conditions for Lactic Acid Recovery Using Organic Base P. Jamal, M. Mel, M.I.A. Karim and N. Syahida International Islamic University Malaysia /Department of Biotechnology Engineering, Faculty of Engineering, K. Lumpur, Malaysia Abstract — Lactic acid has a wide range of applications in food, pharmaceutical and cosmetics industries. The global market for lactic acid in foods is estimated at 275,000 tons with average annual growth of 10 per cent. The persistent demand of pure and naturally produced lactic acid in food and beverage applications attracted researcher’s interest towards process development for maximum recovery of lactic acid from fermentation broth. In this study, an organic base with appropriate diluent was used and optimization of process conditions was done by using central composite design from Statistica software to determine the optimum conditions for recovery of lactic acid. The effects of initial lactic acid concentration, pH, stirring rate, the amount of triisooctylamine in decanol, the ratio of organic phase volume to aqueous phase volume Vorg/Vaq, on the distribution coefficients of the lactic acid, KD, and the percentage extraction of lactic acid were investigated. The optimum process conditions showed maximum recovery of 94.47%, (w/w) and KD value was 17.12. Keywords — Lactic acid, distribution coefficient, optimization, organic base.

I. INTRODUCTION Lactic acid (Į-hydroxypropionic acid) is a chemical compound that plays a role in several biochemical processes. It was first discovered in 1780 from sour milk. Lactic acid is an organic acid with a wide variety of industrial applications in food, pharmaceutical and cosmetics industries [1]. The most important applications are: its use as a preservative and acidulant in foods, as a controlled delivery of drugs in pharmaceutical agents, as a precursor for production of polymer in plastic industries and in leather tanning and textile dyeing [2]. New applications, such as degradable plastics made from poly (lactic) acid, have the potential to greatly expand the market for lactic acid, if more economical processes could be developed [3]. Microbial lactic acid production is gaining importance because it could be produced by fermentation of renewable resources in order to achieve an economical route. Commercial success is not yet achieved due to high product recovery cost and complex nature of the biological process. In the recent years, the demand of pure, naturally produced lactic acid is attracting interest of researchers for development of a better

process which, could contribute towards maximum lactic acid recovery from fermentation broth. A number of approaches can be used for separation of lactic acid from fermented medium, which are extraction by solvents, ion-exchange separation, adsorption, vacuum distillation, membrane separation etc. Various studies have been conducted in finding the most effective and efficient method to extract maximum lactic acid from fermentation broth. The percentage of recovery is still a challenging task due to a complex mixture of components in a fermentation broth. Each of the above mentioned methods exhibits some advantages and disadvantages. Reactive liquid-liquid extraction by a suitable extractant has been found to be a promising alternative to the conventional processes [4, 5]. The choice of the separation process should be based on the efficient and economical usage of the extracting solvent. In recent years, extraction by using aliphatic tertiary and quaternary amines has been studied by several authors due to their properties of forming ion pairs with undissociated carboxylic acid [6, 7]. Many diluents such as oleyl alcohol, chloroform, methyl isobutyl ketone etc. have been screened by researchers because they affect the basicity of the amine, the stability of the acid-amine complex formed and its solvation power [5, 8 ]. These diluents (modifier) help in stabilizing the acid-amine complex because its hydrogen bonds with the oxygen accessible on the acid. Thus, for a reactive extraction, a diluent, which has an acid-interacting functional group, is preferable to non polar aliphatic hydrocarbon [9]. Moreover, pH also exhibits some effect on distribution coefficient, KD [10]. Therefore, in this study, successful efforts were made to optimize the process conditions by considering the effects of initial lactic acid concentration, pH, stirring rate, amount of triisooctyl amine in decanol, and the ratio of organic phase volume to aqueous phase volume Vorg/Vaq, on the distribution coefficients of the lactic acid (KD). The percentage recovery of lactic acid was also investigated. Design of the experiment was selected from STATISTICA 6.0 software in order to determine the optimum process conditions for maximum recovery of lactic acid. Central composite design was used and five levels were considered for all five factors mentioned earlier. Each experiment was conducted three times in order to obtain an average of three replicates.

N.A. Abu Osman, F. Ibrahim, W.A.B. Wan Abas, H.S. Abd Rahman, H.N. Ting (Eds.): Biomed 2008, Proceedings 21, pp. 875–878, 2008 www.springerlink.com © Springer-Verlag Berlin Heidelberg 2008

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P. Jamal, M. Mel, M.I.A. Karim and N. Syahida

E. Experimental design

II. MATERIALS AND METHOD A. Preparation of amine Amine organic solution was prepared by diluting 10-50% triisooctyl amine in decanol. B. Preparation of lactic acid Five different concentrations of aqueous lactic acid solutions were prepared by diluting the lactic acid with distilled water. C. Experimental method Liquid-liquid extractions were performed using the recommended method [11]. Experiments were performed in 250 ml shake-flasks with a working volume of 50 ml in the reciprocal shaker. Each experiment was performed three times and all results were recorded as the calculated mean value. The initial pH of the pure lactic acid solution was adjusted by addition of sodium hydroxide (2M NaOH) or a hydrochloric acid (6M HCl) solution that was measured by Mettler Toledo pH meter. D. Analytical methods Lactic acid concentration in aqueous phase was determined using the YSI 2700 Select Biochemistry Analyzer. The measured lactic acid concentration was used to check the amount of mass transfer of lactic acid from one phase to the other. 1 mL of sample from the aqueous layer was transferred into a cuvette for measuring the lactic acid concentration. Lactic acid concentration was directly measured using Innova YSI 2700 Biochemistry Analyzer with YSI Lactate Membrane. The reaction between the membrane and the sample is as below: L-lactate + O2

L-lactate oxidase

H2O2 + Pyruvate

Distribution coefficient is the equilibrium constant for the distribution of a solute between two immiscible layers [11]. The partitioning of a solute between both layers depends on the affinity of the solute for the organic phase and aqueous phase. The partition coefficient measures the relative affinity of a given compound for two solvents. The calculated distribution coefficient, KD was defined as: KD = lactic acid in organic phase lactic acid in aqueous phase

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Central Composite Design from STATISTICA software was used for optimization of process conditions in order to determine the effect of parameters on each other in a multiparameter system. The total numbers of experiments were 30 with five factors (independent variables) and five levels for each factor plus three center points (all factors at zero level). The independent variables selected were pH, initial lactic acid concentration (g/L), stirring rate (rpm), base concentration (%) and the ratio of organic phase volume to aqueous phase volume (%), and dependent variables (responses) were distribution coefficient and percentage extraction of lactic acid. The levels for all variables and their actual values for optimization are shown in Table 1. STATISTICA 6.0 software (StatSoft) was used for regression and graphical analysis of the data. Regression analysis was done, where multiple regression equation was developed; and it was followed by analysis of the regression equation by statistical analysis. Analysis of variance (ANOVA) and the explanation of variance by the model were given by the multiple coefficient of determination, R2.The significance of the regression coefficients was determined by Student’s t-test. The overall model significance was determined by Fisher’s test and significance of each coefficient was determined by P-values. The variance explained by the model is given by the multiple coefficient of determination, R2. The percentage extraction of lactic acid was taken for analyzing the data using STATISTICA software. A second order polynomial equation was then fitted to the data by multiple regression procedure. For a 5-factor system, the model polynomial equation is given below: Y = ȕ0 + ȕ1 X1 + ȕ2 X2 + ȕ3 X3 + ȕ4 X4 + ȕ5 X5 + ȕ11 X12 + ȕ22 X22 + ȕ33 X32 + ȕ44 X42 + ȕ55 X52 + ȕ12 X1 X2 + ȕ13 X1 X3 + ȕ14 X1 X4 + ȕ15 X1 X5 + ȕ23 X2 X3 + ȕ24 X2 X4 + (1) ȕ25 X2 X5 + ȕ34 X3 X4 + ȕ35 X3 X5 + ȕ45 X4 X5

Table 1 Factors & levels for optimization process condition Factors

Levels -2 5

-1 10

0 15

+1 20

+2 25

1

2

3

4

5

Stirring rate (rpm)

50

100

150

200

250

Concentration of base (%) Ratio of Vorg/Vaq (%)

10

20

30

40

50

20

30

40

50

60

Initial lactic acid concentration (g/L) pH

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Optimization of Process Conditions for Lactic Acid Recovery Using Organic Base

Where Y is the percentage extraction of lactic acid (%, w/w), predicted response; ȕ0, intercept; ȕ1, ȕ2, ȕ3, ȕ4, ȕ5 linear coefficients; ȕ11, ȕ22, ȕ33, ȕ44, ȕ55 squared coefficients; ȕ12, ȕ13, ȕ14, ȕ15, ȕ23, ȕ24, ȕ25, ȕ34, ȕ35, ȕ45 interactions coefficients. III. RESULTS AND DISCUSSION Reactive extraction of lactic acid with triisooctyl amine in decanol was studied by conducting 30 experiments (Table 2). Each experiment was performed three times and the results were taken as the calculated mean value. The statistical analysis of the data was done by the statistical software and the developed polynomial regression equation relating to the lactic acid extraction with the independent variables X1, X2, X3, X4 and X5 is given below: Y (extraction of lactic acid, % (w/w)) = - 14.0 + 0.73 X1 + 11.8 X2+ 0086 X3+ 2.00 X4+ 1.85 X5 + 0.017 X1 X2 0.00149 X1 X3 – 0.0073 X1 X4 + 0.0041 X1 X5 + 0.0009 X2 X3 + 0.098 X2 X4 - 0.012 X2 X5 – 0.00253 X3 X4 – 0.00224 X3 X5 – 0.0133 X4 X5 – 0.0225 X1 X1 - 3.04 X2 X2 + (2) 0.000352 X3 X3 – 0.0165 X4 X4 – 0.00870 X5 X5 From the data obtained, the highest percentage of lactic acid observed was 94.47% (w/w) and KD equals to 17.12, when the highest level of ratio organic/aqueous was used. The process conditions of run 26 can be considered as optimum for reactive extraction of the lactic acid, which are: initial lactic acid concentration of 15 g/L at pH 3 with 30% amine concentration and 60% organic/ aq ratio at 150 rpm stirring rate. The percent recovery of lactic acid obtained was taken as the dependent variables or response Y. The combination of variables and experimental results are presented in Table 2 . The goodness of the model was evaluated using determination coefficient, R2, which was calculated to be 0.907, indicated that 90.7% of the variability of response could be explained by the model [12] or only 9.3% of the total variation was not explained by the model. The closer value of multiple correlation coefficients, R to 1 expressed better correlation existed between the experimental and the predicted value. The value of R is 0.952 indicating good agreement existed between the experimental and predicted values of percentage lactic acid in organic solvent. The p-values along with the second order polynomial coefficient were evaluated using ANOVA. The significance of each coefficient was determined by p-values. The interaction pattern between the variables is indicated by these coefficients. The variables with low probability levels contribute to the model, whereas the others can be neglected and eliminated from the model due to not very significance.

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According to Techapun et al. [13], the low p-value which is less than 0.05, indicates more significant correlation of coefficients. Analysis indicated that as linear model, pH, percent amine and organic ratio are the main factor on extracting lactic acid. The indication is very significant based on the p-value obtained for pH, percent amine and organic ratio as 0.001832, 0.007886 and 0.000465, respectively. It can be concluded that organic ratio is the best factor followed by pH and percent amine. The F-values and p-values for the linear, quadratic and interactive term are given in the Table 2. From this table it is clearly shown that the highly significant parameter is organic ratio (X5) indicated by high F-value 28.58 with lowest probability, p-value 0.000465. Table 2 Experimental design for optimization and results

Run initial LA

pH

RP M

% ami ne

ratio Dis. org/a Coeff. q

X1

X2

X3

X4

X5

1

10

2

100

20

50

2

10

2

100

40

30

3

10

2

200

20

30

4

10

2

200

40

50

5

10

4

100

20

30

6

10

4

100

40

50

7

10

4

200

20

50

8

10

4

200

40

30

9

20

2

100

20

30

10

20

2

100

40

50

11

20

2

200

20

50

12

20

2

200

40

30

13

20

4

100

20

50

14

20

4

100

40

30

15

20

4

200

20

30

16

20

4

200

40

50

17 18

5 25

3 3

150 150

30 30

40 40

19

15

1

150

30

40

20

15

5

150

30

40

21

15

3

50

30

40

22

15

3

250

30

40

23

15

3

150

10

40

24

15

3

150

50

40

25

15

3

150

30

20

26

15

3

150

30

60

27

15

3

150

30

40

28

15

3

150

30

40

29

15

3

150

30

40

30

15

3

150

30

40

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10.0 5.9 5.25 9.47 2.46 9.67 6.25 5.49 3.35 12.16 11.05 5.04 5.51 4.36 3.18 5.85 7.71 5.22 8.32 1.73 11.93 11.2 2.7 9.95 3.1 17.12 10.19 7.93 8.62 9.42

% L.A

% L.A

Obs.

Pred.

90.9

91.53

85.5

87.58

84

83.97

90.5

92.35

71.1

68.8

90.6

90.21

86.2

83.65

84.6

83.57

77

77.09

92.4

94.39

91.7

91.58

83.45

84.79

84.65

82.25

81.35

80.45

76.1

73.05

85.4

84.26

88.6 83.92

89.06 85.61

89.27

84.56

63.4

70.29

92.27

92.49

91.8

93.72

72.93

77.28

90.87

88.6

75.6

77.24

94.47

94.98

91.07

89.58

88.8

89.59

89.6

89.59

90.4

89.59

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P. Jamal, M. Mel, M.I.A. Karim and N. Syahida Table 3 Analysis of Variance (ANOVA)

Factor (1) Initial conc. LA(L) Initia conc. LA(Q) (2) pH (L) pH(Q) (3) stirring rate (L) Stirring rate(Q) (4) % amine(L) % amine(Q) (5) org. ratio(L) org. ratio(Q) 1L by 2L 1L by 3L 1L by4L 1L by 5L 2L by 3L 2L by 4L 2L by 5L 3L by 4L 3L by 5L 4L by 5L Error Total SS

ANOVA; Var.:% recovery; R-sqr=90653; Adj.:69881 5 factors, 1 block,30 Runs; MD Residual=16.6846 SS df MS F P 17.871 1 17.8710 1.07111 0.3277 8.459

1

8.4588

0.50698

0.4944

316.706 247.234 2.354

1 1 1

316.706 247.234 2.3542

18.9819 14.8180 0.1411

0.0018 0.0039 0.7158

20.657

1

20.6565

1.2380

0.2946

192.761

1

192.761

11.5532

0.0078

72.883 476.893

1 1

72.8826 476.893

4.3682 28.5828

0.0661 0.0004

20.178 0.114 2.213 2.139 0.660 0.035 15.308 0.214 25.629 20.138 28.223 150.161

1 1 1 1 1 1 1 1 1 1 1 9

20.1782 0.1139 2.2127 2.1389 0.6602 0.0352 15.3077 0.2139 25.6289 20.1377 28.2227 16.6846

1.2093 0.0068 0.1326 0.1282 0.0359 0.0021 0.9174 0.0128 1.5360 1.2069 1.6915

0.2999 0.9359 0.7241 0.7285 0.8467 0.9643 0.3631 0.9123 0.2465 0.3004 0.2257

1606.47

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V. REFERENCES 1.

2. 3. 4. 5. 6. 7.

8. 9.

IV. CONCLUSION

10.

Carboxylic acids are poorly extractable by common organic solvent due to their hydrophilic nature. Therefore, optimization of process conditions was done by using organic amine for their recovery from aqueous solution. There are reversible complexation reactions between the organic acid and the solvent phase. The proposed method and solvents in this project has successfully recovered 94.47 % lactic acid from aqueous solution through liquid-liquid extraction. Thus maximum amount of lactic acid was recovered by using the optimum operating conditions. The distribution coefficient, KD was 17.12.The important process

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parameters were pH, percentage amine and percentage ratio of organic/aqueous phase, which have significant effect on the percentage extraction of lactic acid into the organic phase.

11. 12.

13.

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