Optimization of Fermentation Medium for Producing α-Hydroxyphenylacetic Acid by Using Plackett-Burman Design and Response Surface Methodology

doi:10.4236/ajac.2013.410070

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American Journal of Analytical Chemistry, 2013, 4, 594-599

http://dx.doi.org/10.4236/ajac.2013.410070 Published Online October 2013 (http://www.scirp.org/journal/ajac)

Optimization of Fermentation Medium for

Producing α-Hydroxyphenylacetic Acid by Using

Plackett-Burman Design and Response Surface

Methodology*

Zhiguo Hou1, Bingmei Chen1, Jing Lan1, Yueman Liu1, Xiaoping Xu1#, James Yu Gu2, Junjie Gu3

1Collage of Chemistry and Chemical Engineering, Fuzhou University, Fujian, Fuzhou, China

2Department of Chemical Engineering, University of Ottawa, Ottawa, Canada

3Department of Mechanical and Aerospace Engineering, Carleton University, Ottawa, Canada

Email: #[email protected]

Received May 9, 2013; revised June 9, 2013; accepted July 10, 2013

which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

ABSTRACT

Plackett-Burman design and response surface methodology were applied in order to optimize the fermentation medium

of (R)-α-hydroxyphenylacetic acid ((R)-HPA) producing Bacillus sp. HZG-19. The factors playing important roles in

the production of (R)-HPA were selected based on Plackett-Burman design. The path of steepest ascent was undertaken

to optimize said fermentation medium. Finally, the optimal levels of the factors with the greatest change in regard to

product yield were further optimized using Box-Behnken and response surface analysis. The optimal conditions were

found to be as follows: casein peptone 30.49 (g × L−1), glycerol 14.09 (g × L−1), KH2PO4 0.1345 (g × L−1), K2HPO4

0.01 (g × L−1), CaCl2 0.1 (g × L−1), MnSO4 0.01 (g × L−1). Under the optimal conditions described above, the yield of

(R)-HPA reached 63.30%, which indicated an increase of 14.9%, as compared to the yield obtained before optimization.

Keywords: α-Hydroxyphenylacetic Acid; Fermentation Medium; Biotransformation; Response Surface Analysis

1. Introduction

α-Hydroxyphenylacetic acid (mandelic acid) (HPA) and

its derivatives are key intermediates for the production of

various pharmaceuticals, such as semi-synthetic penicil-

lins and cephalosporins [1-3]. It is also used as a chiral

resolving agent and chiral synthon for the synthesis of

anti-tumor and anti-obesity agents [4]. Many methods

have been reported for the preparation of enantiomeri-

cally pure (S)-or (R)-α-Hydroxyphenylacetic acid [5-9].

For example, the synthesis of enantiopure α-Hydroxy-

phenylacetic acid has been investigated through dias-

tereomeric crystallization [10]. It has also been prepared

by the chemo-enzymatic routes [11], from methyl man-

delate by lipase-catalyzed hydrolysis [12], or from ki-

netic resolution [13]. However, these approaches are lim-

ited in their industrial application due to the expensive

catalysts, limited efficiency and low yields.

Recently we have reported a new bacterial strain, Ba-

cillus sp. HZG-19, which is capable of degrading phenyl-

glyoxylic acid (PGA) and affording (R)-HPA with high

optical purity. Numerous variables will have an effect on

the production of HPA, hence, factors playing important

roles in the production of pure enantimomeric (R)-HPA

are crucial for large scale production. Plackett-Burman,

Box-Behnken design and response surface methodology

are inexpensive and accurate methods for further opti-

mization of the fermentation medium.

2. Materials and Methods

2.1. Strain and Chemicals

The strain (Bacillus sp. HZG-19) was preserved in the

lab of Chemical Engineering Department, Fuzhou Uni-

versity. (R)-HPA and (S)-HPA were purchased from

Sigma (St. Louis, MO USA). Phenylglyoxylic acid (PGA,

>98%) was supplied by Pharmaceutical & Chemical Co.,

Ltd of Taizhou, China. Methyl alcohol of HPLC grade

was purchased from Merck, Germany. Hydroxypropyl-

*The work was supported by the Doctoral Fund of Ministry of Educa-

tion (No. 20103514110002) and the Major Plan Project of Science and

Technology of Fujian Province (No. 2009N0046).

#Corresponding author.

Z. G. HOU ET AL. 595

β-cyclodextrin (HP-β-CD) was supplied by Fluka (Neu

Ulm, Germany). All other chemicals were obtained from

local suppliers and of reagent grade.

2.2. Medium

Seed medium (g × L−1): casein peptone 10, beef extract 5,

maltose 10, NaCl2, pH 7.2.

Fermentation medium (FM) (g × L−1): casein peptone

20, glycerol 10, K2HPO4 0.01, KH2PO4 0.1, CaCl2 0.1,

MnSO4 0.01, pH 7.2.

Medium for slant culture (SM): same as seed medium

with 2% (w/v) of agar.

2.3. Culture Conditions

The strain, Bacillus sp. HZG-19, was inoculated into 20

ml seed medium in 50 ml Erlenmeyer flasks and cultured

aerobically at 32˚C on a shaker (180 rpm). When the

cells reached logarithmic phase, cultures were then in-

oculated (1%, v/v) into 50 ml flasks containing 20 ml of

FM medium at the same fermentation conditions. PGA

solution, with a final concentration of 15 mM, was added

directly into the fermentation broth under aseptic condi-

tions. After 24 h of incubation, the supernatant that was

obtained after centrifugation from the medium and was

subjected to HPLC analysis and HPCE analysis to de-

termine the concentration and purity of the HPA gener-

ated.

2.4. Analytical Methods

The concentrations of substrate and product were deter-

mined by HPLC using a reverse phase column (Agilent

HC-C 18, Ø 4.6 mm × 250 mm, 5 μm). The mobile phase

is composed of methanol and phosphate buffer (25

mmol/L) (15:85, v/v) containing 6 mmol/L of tetrabu-

tylammonium bromide (pH 6.8) at the rate of 1.0 ml/min.

A UV detector (at 220 nm) was employed for quantifica-

tion.

The yield of HPA is expressed as:





Yield%CHPA CPGA100%,

where CHPA and CPGA represent the concentration of

the HPA generated and initial concentration of PGA,

respectively. The concentration of (R)-HPA and (S)-HPA

were determined by HPCE. Detection was made at 214

nm using a buffer solution of Tris-phosphoric acid (100

mmol/L, pH 7.6) containing 150 g/L of hydroxypropyl-

β-cyclodextrin. A voltage of 20 KV was applied at a

temperature of 20˚C.













Enantiomeric excesse.e.%

RSRS 100%,

(S)-HPA, respectively.

where [R] and [S] are the concentration of (R)-HPA and

3. Results and Discussion

is extremely useful in

ation of experi-

Table 1. Design matrix and experimental results of Plack-

123(X4)X

5(X6) X7 (X8) X9Yield/%

3.1. Plackett-Burman Design

The Plackett-Burman (PB) design

screening and selecting for the most vital factors within a

large candidate pool [14]. The experiments were carried

out according to the design matrix shown in Table 1,

with each row representing one trial while each column

represents a single variable. The six factors listed are:

casein peptone, glycerol, KH2PO4, K2HPO4, CaCl2,

MnSO4, respectively. Three variables (X4, X6, X8) are

dummy variables employed to evaluate the standard er-

rors of the experiment. Elements (1) and (−1) are repre-

sentative of the relative amounts each variable factor,

either high or low, within each experimental trial. Yield

of HPA is considered as response value.

Levels selection and significance evalu

ental variables are summarized in Table 2. Generally,

effects with a confidence level of greater than 95% are

considered to be actual effects, whereas a confidence

level of less than 95% indicates that the effect may have

resulted due to chance. As shown in Table 2, the confi-

dence level of factors casein peptone (X1), glycerol (X2)

and KH2PO4 (X3) are shown to be above 95% and con-

sidered to be significant. The rest of the factors, K2HPO4

(X5), CaCl2 (X7) and MnSO4 (X9) had a confidence of

below 95% in HPA production and hence, were consid-

ered insignificant. The high level of X5, X7 and the low

level of X9 were selected during further optimization

studies.

ett-Burman design.

Run XX X

1 1−11 −1−1−1 1 1 1 59.52

2 11−11 −1−1 −1 1 160.71

3 −11 1 −11 −1 −1 −1 152.83

4 1−111 −11 −1 −1 −1 61.72

5 11−11 1 −1 1 −1 −162.34

6 111−11 1 −1 1 −1 61.31

7 −1111 −11 1 −1 156.63

8 −1−111 1 −1 1 1 −1 54.02

9 −1−1−11 1 1 −1 1 142.12

10 1−1−1−11 1 1 −1 151.32

11 −11−1−1−11 1 1 −1 51.90

12 −1−1−1−1−1−1 −1 −1 −145.05

Eapeafo ilichmoch exerimnt ws perrmedn tripates, te sae belw.

Z. G. HOU ET AL.

596

Table 2. Levels selection and significance evaluation of ex-

perimental variables.

Levels

Code

Low (−gh (1)

T-test Pr > |t| Significancy

1) Hi

X1 20 13.1833 0.0057 1* 30

X2 10 15 7.7648 0.0162 3*

X3 0.1 0.15 7.9100 0.0156 2*

X5 0.01 0.015 −2.8302 0.1054 6

X 0.

0. −

1 0.15 2.9269 0.0996 5

X9 0.01 015 3.2172 0.0845 4

*Ine thatignificanf m5%nce.

eepest ascent search

g/L)glycerol (g/L) KH2PO4 (g/L)Yield (%)

dicat the scy is oore than 9 confide

3.2. Steepest Ascent Design to Approach the

Optimal Region

The steepest ascent method was applied in order to in-

vestigate optimal substrate concentration, by slowly in-

creasing substrates along the path of steepest ascent until

no further increase in response is observed [15]. Based

on the experimental results of PB, casein peptone, glyc-

erol and KH2PO4 should be increased due to their posi-

tive effect (positive value for t). Table 3 represents the

design and results of the steepest ascent search experi-

ment. As shown in Table 3, optimal fermentation me-

dium should be around run 2, so the level of run 2 is con-

sidered as center point in the follow-up response surface

experiment.

3.3. Response Surface Analysis (RSM) and

Establishment of Optimum Fermentation

Medium

RSM is a statistical method by which one can find the

best conditions in a multi-factor system [16]. Among

RSM, Box-Behnken design (BBD) method and central

composite design are more frequently used than the oth-

ers. When the number of the experimental factors does

not exceed 5, Box-Behnken method is more economic.

Casein peptone, glycerol and KH2PO4 were determined

to be the main factors as shown through the PB experi-

ment.

X1, X2, X3 represent casein peptone, glycerol, KH2PO4

respectively and the yield of HPA shows the response

value. Factor levels were determined by steepest ascent

experiment. In order to investigate the optimum levels of

those variables and study their interactions, a three-factor

three-level BBD was applied.

A total of 15 experiments were performed in triplicates,

12 of which used different factors while 3 trials were

used as controls. Controls were used to estimate experi-

mental error. Table 4 illustrates the coded and non-coded

values of the experimental variables, with results of the

BBD experiments given in Table 5.

Table 3. Design and results of the st

experiment.

Runcasein peptone (

1 25 12.5 0.125 54.12

2 28 13.5 0.135 61.92

3 31 14.5 0.145 59.37

4 34 15.5 0.155 54.10

5 37 16.5 0.165 52.35

Table 4. The coded and non-coded values of the experi-

Coded level

mental variables.

Factors Code

−1 0 1

casein peptone (g/L)X1 25 28 31

glycerol (g/L) X2 12.5 13.5 14.5

KH2PO4 (g/L) X3 0.125 0.135 0.145

Table 5. Design and results of BBD experiments.

Run X1 23

X X Yield/%

1 −1 −1 0 55.53

2 −1 1 0 57.04

3 1 −1 0 56.3

4 1 1 0 62.43

5 0 −1 −1 54.13

6 0 −1 1 50.39

7 0 1 −1 56.06

8 0 1 1 56.89

9 −1 0 −1 52.78

10 1 0 −1 58.78

11 −1 0 1 52.68

12 1 0 1 56.42

13 0 0 0 61.83

14 0 0 0 62.02

15 0 0 0 61.93

ased on the experimental results of BBD (Table 5)

d regression analysis, a quadratic polynomial equation

was established to identify the relationship between yield

and variables. The model of coded units can be expressed

as:

132 23

Y61.92667 1.9875X2.00875X

0.67125X1.652083X1.155X X

0.565X X2.449583X1.1425XX

5.109583X





 



(1)

where Y1 is the yield of HPA, X1, X

2 and X3 represent

casein peptone, glycerol and KH2PO4, respectively.

Z. G. HOU ET AL.

597

eter-

was reliable with an R2 value of 0.9913. It suggested that

model was unable to explain only 0.87% of the total

variations. A low value of coefficient of the variation (C.

V.) (1.0298) clearly indicated a very high degree of pre-

cision and a good deal of reliability of the experimental

values.

Analysis of variance (ANOVA) is important in d

ining the adequacy and significance of the quadratic

model. ANOVA summary was shown in Table 6. The F-

value of 63.1957 implies that the model was significant.

A p value of <0.0001 indicated that there was only a

0.01% chance that model’s large F-value could occur due

to noise. Values of “prob > F” less than 0.0500 indicated

the significant model terms. The mathematical model

Figures 1-2 show the response and contour curves for

casein peptone, glycerol and KH2PO4. Contour curves

Figure 1. Response surface plot and contour plot for HPA yield (%) as a function of casein peptone and KH2PO4 (X1 and X3

represent casein peptone and KH2PO4, respectively).

Figure 2. Response surface plot and contour plot for HPA yield (%) as a function of glycerol and KH2PO4 (X2 and X3 repr

sent glycerol and KH2PO4, respectively).

Z. G. HOU ET AL.

598

Table 6. Estimated value of regression equation partial re-

ression coefficient and analysis of squareg deviation.

|t|Term Parameter

estimate Standard error T-test Pr >

X1 1.9875 0.2076 9.5750 0.0002

X2 2.0088 0.2076 9.6774 0.0002

X3 − −

− −

−

DF SS P

196.55 21.8

Quadr 112.

197. 13% CV = 1.0298

0.6713 0.2076 3.2338 0.0231

1 * X−1.6521 0.3055 −5.4071 0.0029

X1 * X2 1.155 0.2936 3.9346 0.0110

X1 * X3 0.565 0.2936 1.9247 0.1122

X2 * X2 2.4496 0.3055 −8.0173 0.0005

X2 * X3 1.1425

−

0.2936 3.8920 0.0115

X3 * X3 5.1096 0.3055 16.7233 0.0001

Source MSF-valuerob > F

Model 9 0478263.19570.0001

Linear 3 67.4865

116.

22.4955 65.26330.0002

atic 3 7248 38.9083 87960.0001

nteraction 3 11.8342 3.9447 11.44440.0112

Error 5 1.7234 0.3447

Total 14 769 R = 99.

rep the fu

f two independent variables with another variable bei

neously it obtained the

logy for production of HPA.

dy clearly indicate that RSM is an ef-

ork was financially supported by the Doctoral

Fund of Ministry of Education (No. 20103514110002)

Science and Technology of

er and P. Lanz, “6-Acyl

Derivatives of Aminopenicillanic Acid,” US Patent No.

3957758, 1976

[2] J. Mills, K. K Shaw, “Phenetha-

doi.org/10.1016/S0040-4039(97)10578-0

resentHPA yield as anction of concentrations

ng [

at a fixed level. Figure 1 shows that HPA yield increases

firstly and decreases slowly afterward with the increase

of casein peptone, and that moderate KH2PO4 results in

high HPA yield. Figure 2 demonstrates that HPA yield

increases firstly and then decreases slowly with decreas-

ing glycerol, whereas moderate KH2PO4 caused HPA

yield increasing. This could be attributed to the fact that

casein peptone, KH2PO4 and glycerol were directly re-

lated with the activity of cell. As seen from Figures 1

and 2, there was a maximum response at the optimum

level of each variable and exist the interaction among the

three variables, so it was not simple linear relationship

for the effect of response value.

The ridge analysis indicated that the regression equa-

tion has no singularity. Simulta

st response surface conditions and the predictive value

of yield from the regression equation. The best theoreti-

cal levels in experiment were: X1 = 0.816, X2 = 0.592

and X3 = −0.045, that is, casein peptone 30.49 g·L−1,

glycerol 14.09 g·L−1 and KH2PO4 0.1345 g·L−1. Pre-

dicted value of yield given these conditions was 63.35%.

Validation under the optimized conditions was per-

formed in a 50 ml Erlenmeyer flask containing 20 ml

reaction medium. The experiments were conducted in

triplicate. Under optimized conditions, HPA yield

achieved in the verification experiment was 63.30%,

which was very close to the value predicted by model

based on BBD (63.35%).

Selective reduction of PGA with Bacillus sp. HZG-19 is

a very promising techno

4. Conclusion

Results of this stu

fective method for the optimization of fermentation me-

dium. Optimum casein peptone, glycerol and KH2PO4

were found to be 30.49, 14.09 and 0.1345 g·L−1, respec-

tively.

5. Acknowledgements

This w

and the Major Plan Project of

Fujian Province (No. 2009N0046).

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