Production of 2,3-Butanediol by Klebsiella Pneumoniae Using ...

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Abstract The production of 2,3-butanediol by Klebsiella pneumoniae from glucose supplemented with different salts was studied. A suitable medium composition ...
Chinese J. Chem. Eng., 14(1) 132—136 (2006)

RESEARCH NOTES

Production of 2,3-Butanediol by Klebsiella Pneumoniae Using Glucose and Ammonium Phosphate QIN Jiayang(秦加阳), XIAO Zijun(肖梓军), MA Cuiqing(马翠卿), XIE Nengzhong(谢能中), LIU Peihai(刘培海) and XU Ping(许平)* State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, China Abstract The production of 2,3-butanediol by Klebsiella pneumoniae from glucose supplemented with different salts was studied. A suitable medium composition was defined by response surface experiments. In a medium containing glucose and (NH4)2HPO4, the strain could convert 137.0g of glucose into 52.4g of 2,3-butanediol and 8.4g of acetoin in shaking flasks. The diol yield amounted to 90% of its theoretical value and the productivity was 1—1.5g·L-1·h-1. In fed-batch fermentation, the yield and productivity of diol were further enhanced by maintaining the pH at 6.0. Up to 92.4g of 2,3-butanediol and 13.1g of acetoin per liter were obtained from 215.0g of glucose per liter. The diol yield reached 98% of its theoretical value and the productivity was up to 2.1g·L-1·h-1. Keywords 2,3-butanediol production, optimization, fed-batch, Klebsiella pneumoniae

1

INTRODUCTION Interest in microbial production of 2,3-butanediol has been increasing recently due to the extensive industrial application of this product. This colorless and odorless liquid with a high boiling point and a low freezing point is a potential valuable fuel additive. Its - heating value is 27.198kJ·g 1, which is quite near the - value of ethanol (29.055kJ·g 1). Besides, condensation of diol to methyl ethyl ketone (MEK) coupled with subsequent hydrogenation yields octane isomers that can be used to produce high-grade aviation fuel. Currently, the manufacturing of 2,3-butanediol is still growing by an annual rate of 4%—7% due to the increased demand for polybutylene terephthalate resin, γ-butyrolactone, spandex, and their precursors[1―5]. In the production of 2,3-butanediol via a mixed acid pathway, acetoin is an intermediate prior to the formation of 2,3-butanediol during fermentation. The metabolic conversion of acetoin to 2,3-butandiol is reversible. The net reactions forming 2,3-butanediol and acetoin from glucose are Glucose butanediol+2CO2+NADH2+2ATP Glucose acetoin+2CO2+2NADH2+2ATP Therefore, on the mass basis, the theoretical yield of - 2,3-butanediol and acetoin from glucose are 0.5g·g 1 -1 and 0.49g·g , respectively. Many investigations have been carried out on microbial production of 2,3-butanediol in the past few

decades, and high-concentration production of 2,3-butanediol have been reported frequently[6―10]. However, some of them used organic substrates as nitrogen sources, and some used new techniques like membrane bioreactor with cell recycle. The former was costly and difficult for downstream processes, while the latter needed some complex equipment, which might be expensive in practical industrial production. The aim of our work was to establish a feasible method for industrial production of 2,3-butanediol. A medium containing glucose and ammonium phosphate was formulated to produce 2,3-butanediol by Klebsiella pneumoniae. With fed-batch fermentation, the yield of 2,3-butanediol plus - acetoin reached 105.4g·L 1.

2 MATERIALS AND METHODS 2.1 Microorganism and media The organism used in this study was Klebsiella pneumoniae CICC 10011 (bought from the China Center of Industrial Culture Collection). The strain was maintained on nutrient agar slants. The slants were incubated at 37℃ for 14h and the fully grown slants were stored at 4℃. The medium for inoculation contained: glucose, - - - 80.0g·L 1; (NH4)2HPO4, 6.0g·L 1; KCl, 1.8g·L 1; EDTA, - - 0.51g·L 1; MgSO4·7H2O, 0.6g·L 1; FeSO4·7H2O, -1 - 0.0225g·L ; ZnSO4·7H2O, 0.0075g·L 1; MnSO4·7H2O,

Received 2005-04-11, accepted 2005-12-13. * To whom correspondence should be addressed. E-mail: [email protected]. cn

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Production of 2,3-Butanediol by Klebsiella Pneumoniae Using Glucose and Ammonium Phosphate -



0.0038g·L 1; citric acid, 0.21g·L 1; sodium citrate, - 0.294g·L 1[11]. A loop of cells was inoculated into 50ml preculture with the above medium in 500-ml flask and incubated for the desired time at 37℃ with shaking at - 200r·min 1 on a reciprocal shaker. The optimum inoculum age was 12h with a dry cell weight (DCW) at about - 8g·L 1. The media was altered to determine the optimal formulation for subsequent experiments, and the best media for the shaking-flask and fed-batch fermentations were determined as described later in Section 3.2. All media were sterilized at 115℃ for 20min. For shaking flasks fermentations, 5ml inoculum was transferred into 100ml preculture with the best medium in each 500-ml flask. Fed-batch fermentation was performed in a 15-L fermentor with a total work- ing volume of 10L at an impeller speed of 300r·min 1 -1 and an aeration rate of l.0L·min . Temperature was controlled at 37℃, and the pH value was maintained at 6.0 by adding KOH or H3PO4. The fermentor was filled with the optimized medium without glucose, and sterilized in situ at 115℃ for 20min. The carbon source was sterilized separately and then added to the fermentor. For inoculation, 500ml inoculum was transferred into the fermentor. At regular time intervals, samples were removed from the fermentor to determine the cell dry weight, glucose and diol concentrations. Analytical methods The optical density (OD) of the culture was assayed using a spectrophotometer (UV-340, Beckman) at 620nm with appropriate dilution. The value of the optical density was converted to dry cell weight (DCW) using a calibration equation (DCW = 0.5923×OD+1.9774). For quick estimation of the amount of glucose during fermentation, a lactate analyzer (YSI model 2700, USA) was used with a glucose oxidase enzyme electrode[12]. Determination of the products was carried out on a Varian gas chromatograph (GC) 3800. The GC was equipped with a flame ionization detector and a 30m capillary column operated with N2 as carrier gas. The temperatures for the GC were as follows: injector temperature, 280℃; detector, 220℃; initial oven tempera- ture, 50℃ for 1min, followed by 10℃·min 1 ramp to 180℃ for a final 10min hold. The sample was firstly

extracted by butyl acetate, and then injected into the gas chromatograph. The concentration of the products was determined by calibration curves. The response surface experiments were analyzed with the software “Statistica for windows, Release 4.5A of StatSoft, Inc”. 3 RESULTS AND DISCUSSION 3.1 Gas chromatogram of 2,3-butanediol and acetoin As shown in Fig.1, two isomers of 2,3-butanediol could be separated obviously (Though there are three stereoisomers of 2,3-butanediol, its dextro-form was not found during fermentation). The main product of glucose fermentation by Klebsiella pneumoniae was m-2,3-butanediol. This was quite different from another 2,3-butanediol production strain, Bacillus polymxa, which mainly produced D-2,3-butanediol[13]. The accumulation of L-2,3-butanediol during our fermentation experiments may be that it was formed from L-acetoin by reduction of diacetyl, which was catalyzed by NADPH-dependent diacetyl reductase, as proposed by Ui et al.[14].

2.2

Figure 1

Gas chromatogram of 2,3-butanediol fermentation

3.2

Culture optimization For response surface experiments[15], Table 1 was constructed to determine the optimal concentration of (NH4)2HPO4 and MgSO4·7H2O, which were the most important factors in the fermentation. Glucose was - increased to 135.0g·L 1; the other ingradients remained at the same levels as those in section 2.1. Results were shown in Fig.2. The analysis suggests the following response surface: 2

Z = −94.514 + 100.37 x + 104.938 y − 0.202 x − 2

0.49 xy − 53.175 y from which the optimal concentration of (NH4)2HPO4 - and MgSO4·7H2O was determined as 24.0g·L 1 and Chinese J. Ch. E. 14(1) 132 (2006)

Chinese J. Ch. E. (Vol. 14, No.1)

134 -

0.88g·L 1, respectively. Table 1

The response surface experiment design x, g·L-1 y, g·L-1 z, g·L-1 (NH4)2HPO4 MgSO4·7H2O production

Block

1

2

1

1

-1

-1

10

0.2

6.1

2

1

1

-1

30

0.2

44.4

3

1

-1

1

10

1.2

27.2

4

1

1

1

30

1.2

55.7

5

1

0

0

20

0.7

62.2

6

1

0

0

20

0.7

62.1

7

2

-1.41

0

5.86

0.7

2.7

8

2

1.41

0

34

0.7

46.4

9

2

0

-1.41

20

0

21.0

10

2

0

1.41

20

1.4

56.4

11

2

0

0

20

0.7

72.1

12

2

0

0

20

0.7

67.0

Figure 3 Graph of growth (DCW), residual glucose (RG) and diol production during fermentation in shaking flasks ● DCW, g·L-1; ▲ RG, g·L-1 concentration, g·L-1: ■ acetoin; ▼ 2,3-butanediol; ★ 2,3-butanediol+acetoin

In the course of 2,3-butanediol fermentation in shaking flasks, 135.0g of glucose in total 10 flasks was transformed into 52.4g of 2,3-butanediol and 8.4g of acetoin by Klebsiella pneumoniae. The diol yield reached 90% of its theoretical value and the produc- - tivity was 1—1.5g·L 1·h 1. 3.4

Figure 2 Effects of (NH4)2HPO4 and MgSO4·7H2O on the diol production (Z =-94.514+10.037x+104.938y-0.202xx- 0.49xy-53.175yy)

In summary, the optimized medium contain: glucose, - - 135.0g·L 1; (NH4)2HPO4, 24g·L 1; MgSO4·7H2O, -1 -1 - 0.88g·L ; KCl, 1.8g·L ; EDTA, 0.51g·L 1; FeSO4·7H2O, - - 0.0225g·L 1; ZnSO4·7H2O, 0.0075g·L 1; MnSO4·7H2O, -1 -1 0.0038g·L ; citric acid, 0.21g·L ; sodium citrate, - 0.294g·L 1. The medium was used for the latter experiments. Shaking flasks fermentation As shown in Fig.3, the production of 2,3-butanediol was a growth-associated phenomenon. The condition for the maximum product formation was approximately the same as that for maximum biomass yield. This was the same as previously reported[16]. In about 50h, all the glucose was utilized. About 5h after that, 2,3-butanediol reached its maximum yield.

Fed-batch fermentation In fed-batch fermentation, the initial glucose con- centration was 135.0g·L 1. During the fermentation, glucose was added to the batch at the time when the consumption of glucose was the most efficient and the - residual glucose concentration was below 30.0g·L 1. Each time 500.0g of solid glucose was added and in total about 1000.0g of glucose was added. In about 50h, up to 92.4g of 2,3-butanediol and 13.1g of acetoin per liter were obtained from 235.0g of glucose per liter with the result shown in Fig.4. The diol yield reached 98% of its theoretical value and the - - productivity was 2.1g·L 1·h 1.

3.3

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Figure 4 Graph of growth (DCW), residual glucose (RG) and diol production during fed-batch fermentation ● DCW; ▲ RG concentration, g·L-1: ■ acetoin; ▼ 2,3-butanediol; ★ acetoin+butanediol

Production of 2,3-Butanediol by Klebsiella Pneumoniae Using Glucose and Ammonium Phosphate Table 2

135

Comparison of cultivation systems for diol production 2,3-Butanediol+Acetoin

Organism

Medium

Method

yield -1

g·L A. aerogenes K. pneumoniae

A. aerogenes

K. pneumoniae

K. oxytoca

P. polymyxa

K. pneumoniae

K. pneumoniae

glucose and urea glucose and yeast extract glucose and (NH4)2SO4 glucose and (NH4)2SO4 glucose and molasses glucose, yeast extract and tryptone glucose and (NH4)2SO4 glucose and (NH4)2HPO4

g·L ·h

-1

-1

g·g

fed-batch

111.0

0.90

0.36

[6]

double fed-batch

113.0

0.94

0.50

[7]

cell recycle

110.0

5.4

0.48

[8]

cell recycle

39.6

9.84

0.43

[20]

fed-batch

102.9

1.1

0.49

[9]

fed-batch

57.3

0.96

0.41

[10]

35.0

3.50

25.0

4.25

0.42

[21]

105.5

2.1

0.49

Our research

continuous

fed-batch

At the end of the fermentation, the residual glu- cose was at 18g·L 1, maybe because the nitrogen resource or other nutrition components were not enough for keeping the activity of the biomass. 3.5

Ref.

productivity -1

Discussion It is known that three enzymes are involved in the 2,3-butanediol pathway[17,18]: α-acetolactate synthase, α-acetolactate decarboxylase, and acetoin (diacetyl) reductase (also called butanediol dehydrogenase). To study the mechanism of these enzymes, an inorganic medium is necessary. Pousen and Stougaard found that the acetolactate synthase was dependent of Mg2+[19]. This was consistent with the important role it played in our medium. (NH4)2HPO4 was used as the main nitrogen source in our medium, but its effect was not only that. (NH4)2SO4 had ever been used to replace it, but the DCW and the diol yield in that fermentation test were quite low. HPO42- might play an important but unknown role. More research in this respect seems necessary. In the present work, from 215.0g of glucose about 92.4g of 2,3-butanediol and 13.1g of acetoin per liter were obtained. The diol yield reached 98% of its - - theoretical value and the productivity was 2.1g·L 1·h 1. Compared with other reports shown in Table 2, the

yields are on the same level, but the productivity of our process is much higher except for that reported by Zeng et al.[8], who used a membrane bioreactor with cell recycle. In comparison with their process, ours is much simpler and less costly. Besides, because the present medium is inorganic, the recovery of the product from the fermentation broth will be more economical. In conclusion, though the yield and the productivity are not the highest, our process is relatively simpler and highly efficient, possibly feasible for industrial production of 2,3-butanediol. REFERENCES 1 2

3

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