World J Microbiol Biotechnol (2012) 28:15631574 DOI 10.

1007/s11274-011-0960-7

ORIGINAL PAPER

Optimization and scale-up of 2,3-butanediol production by Bacillus amyloliquefaciens B10-127

Taowei Yang Xian Zhang Zhiming Rao Shenghui Gu Haifeng Xia Zhenghong Xu

Received: 30 August 2011 / Accepted: 17 November 2011 / Published online: 26 November 2011 Springer Science+Business Media B.V. 2011

Abstract The effects of culture conditions on 2,3butanediol (2,3-BD) production and its possible scale-up have been studied. A newly isolated Bacillus amyloliquefaciens B10-127, belonged to GRAS microorganisms and showed a remarkable 2,3-BD producing potency, was used for this experiment. Corn steep liquor, soybean meal and ammonium citrate were found to be the key factors in the fermentation according to the results obtained from the PlackettBurman experimental design. The optimal concentration range of the three factors was examined by the steepest ascent path, and their optimal concentration were further optimized via response surface methodological approach and determined to be 31.9, 22.0 and 5.58 g/l, respectively. The concentration of the obtained 2,3-BD increased signicantly with optimized medium (62.7 g/l) when compared with unoptimized medium (45.7 g/l) and the 2,3-BD productivity was about 2.4-fold (The fermentation time was shorten from 72 to 42 h). To observe scaleup effects, batch fermentation was carried out at various working volumes. At a working volume of 20.0 l, the nal 2,3-BD concentration and yield were 61.4 and 0.38 g/g at 36 h with a 2,3-BD productivity of 1.71 g/l h. This result shows similar amount of 2,3-BD obtained in lab-scale

fermentation, and it is possible to scale up to larger fermentors without major problems. Keywords Optimization Scale-up 2,3-butanediol Bacillus amyloliquefaciens B10-127

Introduction The bio-based bulk chemicals production from renewable resources has recently attracted increasing attention as it is a green technology and environment-friendly compared with chemical processes (Hermann et al. 2007). Microbial production of 2,3-butanediol (2,3-BD) is one of the examples. Interest in this bioprocess has been increasing recently due to that 2,3-BD has large number of industrial applications and this course would alleviate the dependence on oil supply for the production of platform chemicals. As an important starting material, 2,3-BD can be used to produce valuable derivatives such as methyl ethyl ketone and 1,3-butadiene (Haveren et al. 2008; Tran and Chambers 1987). Besides, 2,3-BD has wide application in transport fuels production, in the manufacturing of printing inks, perfumes, and fumigants, moistening and softening agents, explosives and plasticizes, pharmaceutical carriers (Celinska and Grajek 2009; Syu 2001). 2,3-BD could be produced from carbohydrates via the mixed acid fermentation pathway by many bacterial species such as Klebsiella pneumoniae (Yu and Saddler 1983), Enterobacter aerogenes (Zeng et al. 1990), Bacillus polymyxa (De Mas et al. 1988) and Serratia marcescens (Neish et al. 1947; Zhang et al. 2010a, b). So far, the most efcient 2,3-BD producers reported are K. pneumoniae, Klebsiella oxytoca and E. aerogenes (Celinska and Grajek 2009). Among all these strains, Bacillus amyloliquefaciens was

T. Yang X. Zhang Z. Rao (&) S. Gu H. Xia The Key Laboratory of Industrial Biotechnology, Ministry of Education, Laboratory of Applied Microorganisms and Metabolic Engineering, School of Biotechnology, Jiangnan University, Wuxi 214122, Jiangsu Province, Peoples Republic of China e-mail: raozm@yahoo.com.cn Z. Xu Laboratory of Pharmaceutical Engineering, School of Medicine and Pharmaceutics, Jiangnan University, Wuxi 214122, Jiangsu Province, Peoples Republic of China

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rarely reported as a producing strain for 2,3-BD production compared with other organisms. While the strain generally recognized as safe (GRAS) microorganism used for industrial production was much safer than 2,3-BD-producing strain K. pneumoniae that cause disease. Optimization of culture medium is a very important aspect in the eld of food microbiology and fermentation to improve product yield and reduce process variability, as well as reducing development time and overall costs (Kennedy and Krouse 1999; Mu et al. 2009). Many researchers are therefore studying processes for the production of 2,3-BD. In the previous study, several strategies have been widely used to enhance 2,3-BD production, such as optimizing medium component, optimizing fermentation operating conditions and establishing mathematical models (Alam et al. 1990; Celinska and Grajek 2009; Ghosh and Swaminathan 2003; Zeng et al. 1994). Nutritional and physiological factors such as media composition, aeration, pH, and temperature of the cell culture are essential for operating fermentative processes. The optimization of environmental and nutritional conditions for production of 2,3-BD by S. marcescens, K. pneumoniae and K. oxytoca have been studied (Zhang et al. 2010a, b), respectively, and the concentration of production showed a considerable improvement under the optimal situation. In our previous work, a GRAS strain B. amyloliquefaciens B10-127, capable of producing 2,3-BD effectively and tolerating glucose up to 300 g/l was isolated. And we also optimized fermentation operating conditions (Yang et al. 2011). Compared with other known 2,3-BD producing strains, the 2,3-BD productivity of this microorganism was not satisfactory, but B. amyloliquefaciens B10-127 is superior for its GRAS status that meets safety regulations for industrial-scale fermentation. In this report, for optimal production of 2,3-BD, physiological and nutritional factors have to be studied; however, scale-up for industrial application production by Bacillus genus has rarely been studied. In the present investigation, the newly developed GRAS strain B. amyloliquefaciens B10-127 was used. Medium optimization was performed using Plackett-Burman and central composite design to maximize the production of 2,3-BD. In order to observe scale-up effects, different sizes of fermentation culture have been tested.

Media and culture conditions The strain B. amyloliquefaciens B10-127 was maintained on agar slants containing the following medium: glucose 60 g/l, peptone 10 g/l, yeast extract 5 g/l, NaCl 5 g/l, and 2% agar at pH 6.5. The slants were incubated at 37C for 14 h, maintained at 4C and subcultured at 4-week intervals. The seed culture was prepared by inoculating a full loop of cells from freshly prepared slants into 50 ml of the following medium: glucose 60 g/l, K2HPO4 4 g/l, yeast extract 5 g/l, corn steep liquor 10 g/l, pH 6.5. The cultivation was conducted in 250-ml shake asks for 10 h with agitation (160 rpm, reciprocal shaker) at 37C. Shaking ask effects of fermentation were investigated in 250 ml Erlenmeyer asks containing 50 ml of fermentation medium. The size of inoculum was 4% (v/v). The basic fermentation medium before optimization contained (g/l): glucose 150 g/l, K2HPO4 6 g/l, corn steep liquor 10 g/l, yeast extract 10 g/l, pH 6.5. The cultures were incubated at 37C on a rotary shaker at 160 rpm. Effect of nitrogen sources on 2,3-BD production The impact of various nitrogen sources on cell growth and 2,3-BD production were rstly investigated by a traditional step-by-step replacing experimental procedure. Sources of nitrogen include organic nitrogen and inorganic nitrogen. The sources chosen for the study were beef extract, yeast extract, peptone, soybean meal, corn steep liquor, ammonium critrate, urea, (NH4)2SO4, (NH4)2HPO4, NH4Cl. Selection of signicant variables by PlackettBurman design The PlackettBurman (PB) design, an efcient technique for medium-component optimization (Plackett and Burman 1946), was used to select signicantly variables for 2,3-BD production, and insignicant ones were eliminated to obtain a smaller, more manageable set of factors. The tted rst-order model is: X Y b0 bi Xi Y is the predicted response, b0 and bi are constant coefcients, and Xi is the coded independent factors. Each variable is represented at two levels, high and low, which are denoted by (?1) and (-1), respectively. A total of eight parameters were included for selection, Table 2 illustrates the levels of each variable used in the experimental design.

Materials and methods Bacterial identication 16S rRNA gene sequence was amplied according to standard procedures (Sambrook and Russell 2001) and compared to the sequences in the GenBank database through BLAST sequence analysis (Yang et al. 2011).

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Path of steepest ascent The method of steepest ascent is a procedure for moving sequentially along the direction of the maximum increase in the response (Box et al. 1978). The direction of steepest ascent is the direction in which 2,3-BD increased rapidly by increasing or decreasing the condition of the signicant factors. Based on the results of the PB experimental design, the optimal level scope of each selected factor was examined by means of this well-known path of steepest ascent method. The path of steepest ascent was initiated from the center of the PB design. Experiments were performed along the steepest ascent path until the response did not increase any more. Central composite designs and response surface analysis The next step in the formulation of the medium was to determine the optimum levels of signicant variables for 2,3-BD production. For this purpose, response surface methodology (RSM) based on central composite design (CCD) with ve coded levels was employed to determine the most signicant factors screened by PB design for enhancing 2,3-BD production (Abdul Rahman et al. 2011; Kennedy and Krouse 1999; Malinowska et al. 2009). The three independent factors were investigated at ve different levels (-1.682, -1, 0, ?1, ?1.682) and the experimental design used for study are all shown in Table 6. The response values (Y) in each trial were the average of the duplicates. The data obtained from RSM on 2,3-BD production were subjected analysis of variance (ANOVA). The experimental results of RSM were tted via the response surface regression procedure, using the following second-order polynomial equation: X X X Y b0 bi xi bii x2 bij xi xj i in which Y is the predicted response, xi and xj are independent variables, b0 is the intercept, bi is the linear coefcient, bii is the ith quadratic coefcient, and bij is the ijth interaction coefcient. Design-Expert, Version 7.0 (STAT-EASE Inc., Minneapolis, USA) was used for the experimental designs and statistical analysis of the experimental data. The analysis of variance (ANOVA) was used to estimate the statistical parameters. Scale-up production of 2,3-BD In order to obtain scale-up factors, batch fermentation experiments were performed at various working volumes using the optimal culture conditions obtained from the

labscale tests. The working volumes were 3.0, 7.0 and 20.0 in 5, 10 and 30 l vessels, respectively (manufactured by Biotron). Optimization of nutritional conditions determined by ask experiments were used for scale-up tests. Agitation speed, aeration rate and temperature were maintained at 350 rpm, 0.66 vvm and 37C which detected in our preliminary tests. Analytical methods The cell mass concentration was determined by measuring the OD at 600 nm in a UV-visible spectroscopy system (UV-2000, UNICO, China). The cell dry weight (DCW) was calculated from the optical density using calibration curve for the strain. The composition of fermentation broth was analyzed using a Agilent 1200 high performance liquid chromatograph (HPLC) system (Agilent Corp., USA) with a RID-10A refractive index detector. The stationary and mobile phases were a Waters SugarPak1 (6.5 mmid 9 300 mm; Waters, USA) and ddH2O at 0.5 ml/min, respectively. The column temperature was controlled at 30C. All experiments were repeated at least three times.

Results Bacterial identication The bacterium used in this study was isolated from our preliminary work. After puried several times, the isolate B10-127 was identied through BLAST analysis of the partial sequences of 16S rRNA gene. It was 99% identical with some sequence of B. amyloliquefaciens according to its 16S rDNA sequence. The sequence was deposited in the GenBank database with accession no. HQ005359. Based on these results, strain B10-127 was identied as a strain of B. amyloliquefaciens and designated as B. amyloliquefaciens B10-127 (Yang et al. 2011). Effect of nitrogen sources on 2,3-BD production The inuences of organic and inorganic nitrogen sources on cell growth and 2,3-BD production were tested. The results (Fig. 1; Table 1) had clearly shown that cell mass and 2,3-BD production were markedly affected by the addition of nitrogen sources. Among the organic nitrogen sources investigated, soybean meal resulted in the maximum 2,3-BD, followed by corn steep liquor (Fig. 1), whereas ammonium critrate proved to be the best among the inorganic compounds tested (Table 1). So soybean meal, corn steep liquor and ammonium critrate were chosen as nitrogen sources for further optimization.

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1566 Fig. 1 Effect of organic nitrogen sources on 2,3-BD production. Beef extract (lled square), Corn steep liquor (lled circle), Soybean meal (lled triangle), Peptone (lled inverted triangle), Yeast extract (lled star)

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Table 1 Effect of Inorganic nitrogen sources on 2,3-BD production Control Urea Ammonium citrate (NH4)2SO4 (NH4)2HPO4 NH4Cl

Time (h) 84 78 66 78 72 84

DCW (g/l) 16.1 17.9 19.1 20.4 19.6 15.7

Acetoin (g/l) 7.8 6.9 4.6 6.3 5.4 8.9

2,3-BD (g/l) 52.1 54.3 57.2 53.9 55.3 50.7

Yield (g/g Glucose) 0.35 0.36 0.38 0.36 0.37 0.34

Productivity (g/l h) 0.62 0.70 0.87 0.69 0.77 0.60

Selection of signicant variables by PlackettBurman design The PB design is a powerful method for screening significant factors. The statistical technique is widely used as a tool for checking the efciency of several processes. To optimize the culture medium in the fermentation of glucose by B. amyloliquefaciens B10-127 to yield 2,3-BD, the constituents of the medium were rstly examined. PB design for a total of eight variables was used to identify which variables have signicant effects on 2,3-BD production (Table 2). The medium includes phosphate, succinic acid, Fe2?, Mn2?, and Mg2?, which signicantly
Table 2 The PlackettBurman design for screening variables in 2,3BD production Factors (g/l) Corn steep liquor Soybean meal Ammonium Citrate K2HPO4 FeSO47H2O MnSO47H2O MgSO47H2O Succinic acid Variables X1 X2 X3 X4 X5 X6 X7 X8 Low level (-1) 10 4 1 1 0 0 0.2 0.1 High level (?1) 20 10 4 4 0.1 0.1 0.6 0.5

affect 2,3-BD production (Garg and Jain 1995; Syu 2001). In addition to these factors, the medium also contained glucose as carbon source and corn steep liquor, soybean meal and ammonium citrate as nitrogen source according to the pre-experiments. The upper and lower limits of each variable were chosen according to the preliminary investigation of the limits of the variables. The PB experimental design for 14 trials with two levels for each variable and the effect of 8 variables on 2,3-BD production are demonstrated in Table 3. To approach the neighborhood of the optimum response, the tted rst-order model equation for 2,3-BD production was obtained from the PB design experiments: Y 53:88 3:63 X1 2:74 X2 5:56 X3 0:17 X4 2:01 X5 0:46 X6 0:56 X7 2:09 X8 Statistical testing was carried out using Fishers test for ANOVA according to the experimental data. The coefcient R2 of the rst-order model was 0.964, indicating that nearly 97% of the variability in the response could be explained by the model. The value of the adjusted determination coefcient (Adj R2 = 89.3%) was also very high to advocate for a high signicance of the model. R2 value of this model higher than 0.9 was considered as having a very high correlation. So it was reasonable to use the regression model to analyze the

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World J Microbiol Biotechnol (2012) 28:15631574 Table 3 PlackettBurman design for screening of signicant factors affecting 2,3-BD production

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Run

Variable levels X1 X2 -1 1 -1 1 -1 1 1 1 0 -1 -1 0 1 -1 X3 1 1 -1 -1 -1 1 1 -1 0 -1 1 0 -1 1 X4 1 -1 -1 1 -1 1 -1 -1 0 1 -1 0 1 1 X5 -1 -1 -1 1 1 -1 1 -1 0 -1 1 0 1 1 X6 1 -1 -1 1 -1 -1 1 1 0 1 1 0 -1 -1 X7 1 1 -1 -1 1 -1 1 -1 0 1 -1 0 1 -1 X8 1 -1 -1 -1 1 1 -1 1 0 -1 1 0 1 -1

2,3-BD (g/l)

1 2 3 4 5 6 7 8 9 10 11 12 13 14

1 1 -1 1 1 -1 -1 1 0 -1 -1 0 -1 1

58.3 60.2 36.3 53.2 52.7 58.8 59.2 58.2 60.4 39.4 57.7 59.6 50.1 62.4

trends in the responses. In this model, the F test and P values were used to identify the effect of each factor on 2,3-BD production. P value below 0.05 indicates that the model terms are signicant. Table 4 shows the effects of the variables on the response and the signicant levels. Based on the statistical analysis, the factors (P \ 0.05) such as X1 (corn steep liquor), X2 (soybean meal), X3 (ammonium citrate) and X8 (succinic acid) had the greatest positive impacts on the production of 2,3-BD, but compared with X1, X2 and X3, X8 had less positive effect. X5 (FeSO4) and X6 (MnSO4) were set at their high levels according to the positive effects although they were nonsignicant to 2,3-BD production. Factors such as X4 (K2HPO4) and X6 (MgSO4) had negative effects and were set at their low levels. And then, corn steep liquor, soybean meal and ammonium citrate were selected for further optimization to obtain a maximum response.

Path of steepest ascent The path of steepest ascent was determined to nd the proper direction of changing variables by increasing or decreasing the value of the main factors. The above results indicated that corn steep liquor, soybean meal and ammonium citrate can signicantly inuenced the 2,3-BD production compared with other factors. To search the proper direction of these three factors with the other factors xed at zero level, the path of the steepest ascent was employed. The design and responses of the steepest ascent experiment are shown in Table 5. It is shown that the highest response was 61.43 g/l when the concentration of corn steep liquor, soybean meal and ammonium citrate was selected to be 30, 20 and 5 g/l, respectively. It suggested that this point was near the optimal point and this combination was used as the middle point for the second-order experiment, i.e., CCD.

Table 4 Effects and statistical analysis of variables Variable Intercept X1 X2 X3 X4 X5 X6 X7 X8 Coefcient 53.88 3.63 2.74 5.56 -0.17 2.01 0.46 -0.56 2.09 Standard error 0.75 0.75 0.75 0.75 0.75 0.75 0.75 0.75 0.75 F value 13.52 23.48 13.43 55.21 0.055 7.21 0.38 0.56 7.82 P value 0.0118* 0.0084* 0.0215* 0.0018* 0.8265 0.0550 0.5732 0.4969 0.0498 Origin 1 2 3 4 5 15 20 25 30 35 40 8 12 16 20 24 28 2 3 4 5 6 7 35.5 47.4 53.2 61.4 58.5 50.9 Table 5 Experiment design and results of the steepest ascent path Run Corn steep liquor (g/l) Soybean meal (g/l) Ammonium Citrate (g/l) 2,3-BD (g/l)

R2 = 0.9643, R2 (Adj) = 0.8930 * Signicant at 99% condence degree (P \ 0.05)

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1568 Table 6 The results of the central composition experiment Run Coded variable level X1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 -1 1 -1 1 -1 1 -1 1 -1.68 1.68 0 0 0 0 0 0 0 0 0 0 X2 -1 -1 1 1 -1 -1 1 1 0 0 -1.68 1.68 0 0 0 0 0 0 0 0 X3 -1 -1 -1 -1 1 1 1 1 0 0 0 0 -1.68 1.68 0 0 0 0 0 0 Real variable level Corn steep liquor (g/l) 25 35 25 35 25 35 25 35 21.6 38.4 30 30 30 30 30 30 30 30 30 30

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Soybean meal (g/l) 16 16 24 24 16 16 24 24 20 20 13.3 26.7 20 20 20 20 20 20 20 20

Ammonium Citrate (g/l) 4.0 4.0 4.0 4.0 6.0 6.0 6.0 6.0 5.0 5.0 5.0 5.0 3.32 6.68 5.0 5.0 5.0 5.0 5.0 5.0

2,3-BD (g/l)

49.1 52.3 54.2 55.5 52.7 57.9 59.7 61.5 53.5 59.8 52.1 59.7 51.6 59.3 61.7 62.3 61.9 62.0 61.5 62.1

Central composite designs and response surface analysis Response surface optimization is more advantageous than the traditional single parameter optimization in that it saves time, space and raw materials (Ryad et al. 2010). Based on the PB design and the path of steepest ascent, RSM using CCD was applied to determine the optimal levels of the three selected variables (corn steep liquor, soybean meal and ammonium citrate) which signicantly inuenced the 2,3-BD production. A total of 20 runs were needed for optimizing the three individual parameters in the current CCD (Table 6). By applying multiple regression analysis on the experimental data, the response variable and the test variables were related by the following quadratic equation: Y 61:93 1:62 X1 2:32 X2 2:46 X3 0:66 X1 X2 0:31 X1 X3 0:29 X2 X3 1:92 X2 2:19 X2 1 2 2:35 X2 3 where Y is the predicted 2,3-BD production (g/l); X1, X2 and X3 are the coded values of corn steep liquor, soybean meal and ammonium citrate, respectively. On the basis of the experimental values, statistical testing was carried out using Fishers test for ANOVA (Table 7). The Students F test and P values were used as a

Table 7 Signicance test of regression coefcient Variable Intercept X1 X2 X3 X1X2 X1X3 X2X3 X2 1 X2 2 X2 3 Model Lack of t R2 = 0.9948, R2 (Adj) = 0.9902 Coefcient 61.93 1.62 2.32 2.46 -0.66 0.31 0.29 -1.92 -2.19 -2.35 Standard error 0.18 0.12 0.12 0.12 0.15 0.15 0.15 0.11 0.11 0.11 188.55 387.65 437.31 18.52 4.12 3.49 280.46 363.23 418.03 214.15 3.64 \0.0001 \0.0001 \0.0001 0.0016 0.0698 0.0914 \0.0001 \0.0001 \0.0001 \0.0001 0.0911 F value P value

tool to check the signicance of each coefcient, which also indicated the interaction strength between each independent variable. For any of the terms in the model, a large regression coefcient and a small P value would indicate a more signicant effect on the respective response variables (Elibol 2004). Thus, the smaller was the values of P, the more signicant was the corresponding coefcient. As

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shown in Table 7, the F and P values were 214.15 and \0.0001, respectively. So the test model was statistically signicant at the 99% level of signicance. The regression equation obtained from the ANOVA showed that the multiple correlation coefcient R2 was 0.9984 (a value [0.75 indicates tness of the model). This is an estimate of the fraction of overall variation in the data accounted by the model, and thus the model is capable of explaining 99.84% of the variation in response. The adjusted R2 is 0.9902 and the predicted R2 is 0.9665, which indicates that the model is good (for a good statistical model, The closer the R2 value is to 1.00, the stronger the model is and the better it predicts the response). Adeq Precision measures the signal to noise ratio. The adequate precision value of the present model was 41.950, and this also suggests that the model can be used to navigate the design space. The adequate precision value is an index of the signal-to-noise ratio, and values of higher than 4 are essential prerequisites for a model to be a good t. The response surface curves are plotted to explain the interaction of the variables and to determine the optimum level of each variable for maximum response. The response surface curves are shown in Figs. 2, 3 and 4. Each gure demonstrates the effect of two factors while the other factors were xed at zero level. The model predicted the optimal values of the three most signicant variables were X1 = 0.38, X2 = 0.51 and X3 = 0.58. Correspondingly, the values of corn steep liquor, soybean meal and ammonium citrate were 31.9, 22.0 and 5.58 g/l, respectively. The maximum predicted concentration of 2,3-BD was 63.5 g/l. By optimization of culture conditions, 2,3-BD production was enhanced from 45.7 to 63.5 g/l, and the fermentation time was shorten from 72 to 42 h.

Fig. 2 Response surface gure (a) and corresponding contour (b) of the mutual effects of corn steep liquor and soybean meal on 2,3-BD production

Scale-up production of 2,3-BD Validation of the second-order polynomial equation Based on the results of medium optimization, the optimum composition for 2,3-BD production by B. amyloliquefaciens B10-127 is as follows (g/l): glucose 150, corn steep liquor 31.9, soybean meal 22.0, ammonium citrate 5.58, K2HPO4 2.5, MgSO47H2O 0.3, MnSO47H2O 0.05, FeSO47H2O 0.05, Succinic acid 0.3. To validate the adequacy of the model equation for predicting maximum 2,3-BD production, three additional experiments in shake asks were performed using the predicted culture conditions. Under the optimized condition, the 2,3-BD average yield of 62.7 g/l was obtained at 42 h, which was obviously in good agreement with the model predicted maximum value of 63.5 g/l. Therefore, this result indicated that the optimized medium favored the production of 2,3-BD. The maximum 2,3-BD concentration of 62.7 g/l at 42 h with a 2,3-BD productivity of 1.49 g/l h was obtained by batch culture in shake asks, although these results were new records on 2,3-BD fermentation by GRAS microorganism to our knowledge, it was less efcient than that of reported high-producers (such as K. pneumoniae, K. oxytoca and E. aerogenes). In order to determine the scale-up capacity, fermentation was carried out at 5-, 10and 30-l fermenter with working volumes at 3.0, 7.0 and 20.0 l, respectively. Optimal media and culture conditions determined by ask experiments were used for scale-up tests. The fermentation results are shown in Fig. 5, when the working volume was increased from 3.0 to 20 l, 2,3-BD production was slightly reduced and the fermentation time was extended by about 4 h. However, the cellular growth

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Fig. 3 Response surface gure (a) and corresponding contour (b) of the mutual effects of corn steep liquor and ammonium citrate on 2,3BD production

Fig. 4 Response surface gure (a) and corresponding contour (b) of the mutual effects of soybean meal and ammonium citrate on 2,3-BD production

and 2,3-BD secretion were similar at the different working volumes. It was also found that 2,3-BD production rapidly reduced after reaching the maximum value and a concomitant increase of acetoin production appeared after the glucose was depleted. The similar phenomenon was also found by Zhang et al. (2011).

Discussion In order to make the production of 2,3-BD economical on industrial scale, high concentration and productivity and low cost of fermentation are essential (Garg and Jain 1995;

Ma et al. 2009). To achieve this aim several strategies have been used such as screening a productive strain, optimizing fermentation operating conditions and establishing mathematical models, beside the mentioned methods, designing an appropriate fermentation medium is also crucial important to improve the efciency and productivity of the fermentation process because product concentration, yield, and cell growth conditions are strongly inuenced by medium composition such as the carbon source, nitrogen source, inorganic salts, and so on (Garg and Jain 1995; Ma et al. 2009). However, there is no general dened medium for 2,3-BD production by different microbial strains because every microorganism has its own special nutritional requirements depending on its environment. So it is

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World J Microbiol Biotechnol (2012) 28:15631574 Fig. 5 Batch fermentation proles of Bacillus amyloliquefaciens B10-127 for the scale-up experiment, determined using various working volumes: a 3.0 l, b 7.0 l, c 20.0 l. Acetoin (lled square), 2,3-BD (lled circle), acetoin ?2,3-BD (lled triangle), DCW (lled inverted triangle), Glucose (lled star)

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not an easy task to explore a medium that contains all the main nutritional factors and obtain their optimum levels. This work primarily aimed at optimizing the process variables for production of 2,3-BD in using statistical optimization technique for multivariable effect. Culture conditions and media composition optimization by a conventional one-at-the-approach led to a substantial increase in 2,3-butanediol yield. However, this approach not only is generally time consuming and requires a large number of experiments to be carried out but also has the limitation of ignoring the importance of interaction of various parameters (Anvari and Safari Motlagh 2011). More efcient analytical techniques are based on RSM (Bezerra et al. 2008). This was rst proposed by Box and his collaborators in 1951 as a method to determine the optimal conditions which maximize or minimize a response (Box and Wilson 1951). It enables a large amount of data to be obtained from a reduced number of experiments, including the potential interactions between the studied factors (Bas and BoyacI 2007). The RSM can be dened as a group of statistical and technical tools used to study the relationship between a response of interest and several input variables. The model has to describe the behavior of a group of data with a view to making statistical predictions. The aim is the simultaneous optimization of several factors to lead to the best performance of a particular system (Bezerra et al. 2008; Ryad et al. 2010). Production of 2,3-BD has been shown to be sensitive to repression by different nitrogen sources (Ma et al. 2009; Zhang et al. 2010a, b). So nitrogen sources were rstly tested on the growth and 2,3-BD production of B. amyloliquefaciens B10-127 by the conventional one-at-theapproach. In the present investigation, results obtained showed that soybean meal, corn steep liquor and ammonium critrate resulted in a positive effect on 2,3-BD production compared to other nitrogen sources. Then statistically based experimental designs proved to be a valuable tool in optimizing the medium for 2,3-BD production by the isolated strain B. amyloliquefaciens B10127. Among the eight variables tested by PB experiments, soybean meal, corn steep liquor and ammonium critrate were identied as the most important components for 2,3BD production. Their optimal concentrations were obtained by using statistical analysis of RSM. In microbial fermentations, the production costs are mainly dependent on the nitrogen source cost (Karin and Barbel 2000), as well as the carbon source cost. The maximum 2,3-BD yield was usually obtained when cells were grown in glucose media containing peptone and beef extract (Alam et al. 1990; Nilegaonkar et al. 1992) or yeast extract (Perego et al. 2000; Zhang et al. 2010a, b). Soybean meal, the by-product after extracting most of the oil from soybeans, which is rich in protein and energy, is an

inexpensive valuable nutrient source available on a large scale. So soybean meal is an inexpensive valuable and stimulating media component for 2,3-BD fermentation. It has an obvious economic advantage as compared to other organic nitrogen sources including yeast extract and peptone. So soybean meal was demonstrated as a good nitrogen source for large-scale 2,3-BD fermentation. Corn steep liquor is a major byproduct of the corn wet-milling industry, contains approximately 47% crude protein and is a low-cost nutrient source available on a large scale (Parekh et al. 1999). Corn steep liquor is widely used in the fermentation industry to produce a variety of substances, such as lactic acid by Streptomyces sp. (Rivas et al. 2004) and ethanol by Zymomonas mobilis (Silveira et al. 2001). The preliminary experiment results have clearly shown that 2,3-BD production was markedly enhanced by corn steep liquor compared with other nitrogen sources culture (Tables 1, 4). This result is in agreement with Ma (Ma et al. 2009). Whats more, soybean meal and corn steep liquor are alternative, low-cost, and high-yield media component for 2,3-BD fermentation and has an obvious economic advantage. Ammonium critrate is also a most important factor that inuenced the 2,3-BD accumulation in this study (Table 4). It is reported that 2,3-butanediol production can be increased by addition of different organic acids, because they are intermediate metabolites for 2,3-BD production (Anvari and Safari Motlagh 2011; Yu and Saddler 1982). So ammonium critrate offers the nitrogen source for cell growth and is also very important to product formation. The scale-up experiment was performed to observe scaleup effects for application in industrial processes. As shown in Fig. 5 there was a slight reduction on 2,3-BD production at a larger working volume. This phenomenon was also nely explained by Oh (Oh et al. 2009). When scale-up experiments were performed, parameters such as temperature and pH were difcult to maintain at the same value, especially at all locations within the fermentor. As the absolute quantity of carbon and nitrogen sources increased, the production of organic acids and endotoxins was increased. The surrounding conditions were thus harmful to cell growth. Therefore, when working volumes increased, cell and production concentration were decreased. And the maximum 2,3-BD concentration was about 67.6 g/l at stationary phase. This result shows similar amount of 2,3-BD obtained in lab-scale fermentation, and it is possible to scale up to larger fermentors without major problems. In conclusion, we conducted a sequential statistical experimental design to optimize the medium for 2,3-BD production by B. amyloliquefaciens B10-127. Corn steep liquor, soybean meal and ammonium citrate had a signicantly effect on 2,3-BD production and the optimal values of the three key factors were 31.9, 22.0 and 5.58 g/l,

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1573 Celinska E, Grajek W (2009) Biotechnological production of 2, 3-butanediolcurrent state and prospects. Biotechnol Adv 27:715725. doi:10.1016/j.biotechadv.2009.05.002 De Mas C, Jansen NB, Tsao GT (1988) Production of optically active 2, 3-butanediol by Bacillus polymyxa. Biotechnol Bioeng 31:366377. doi:10.1002/bit.260310413 Elibol M (2004) Optimization of medium composition for actinorhodin production by Streptomyces coelicolor A3(2) with response surface methodology. Process Biochem 39:10571062. doi: 10.1016/s0032-9592(03)00232-2 Garg SK, Jain A (1995) Fermentative production of 2, 3-butanediol: a review. Bioresour Technol 51:103109. doi:10.1016/09608524(94)00136-O Ghosh S, Swaminathan T (2003) Optimization of process variables for the extractive fermentation of 2, 3-butanediol by Klebsiella oxytoca in aqueous two-phase system using response surface methodology. Chem Biochem Eng Q 17:319325 Haveren Jv, Scott EL, Sanders J (2008) Bulk chemicals from biomass. Biofuels Bioprod Bioref 2:4157. doi:10.1002/bbb.43 Hermann BG, Blok K, Patel MK (2007) Producing bio-based bulk chemicals using industrial biotechnology saves energy and combats climate change. Environ Sci Technol 41:79157921. doi:10.1021/es062559q Karin H, Barbel HH (2000) Factors affecting the fermentative lactic acid production from renewable resources. Enzyme Microb Technol 26:87107. doi:10.1016/S0141-0229(99)00155-6 Kennedy M, Krouse D (1999) Strategies for improving fermentation medium performance: a review. J Ind Microbiol Biotechnol 23:456475. doi:10.1038/sj.jim.2900755 Ma C, Wang A, Qin J, Li L, Ai X, Jiang T, Tang H, Xu P (2009) Enhanced 2, 3-butanediol production by Klebsiella pneumoniae SDM. Appl Microbiol Biotechnol 82:4957. doi:10.1007/ s00253-008-1732-7 Malinowska E, Krzyczkowski W, Lapienis G, Herold F (2009) Improved simultaneous production of mycelial biomass and polysaccharides by submerged culture of Hericium erinaceum: optimization using a central composite rotatable design (CCRD). J Ind Microbiol Biotechnol 36:15131527. doi:10.1007/s10295009-0640-x Mu W, Chen C, Li X, Zhang T, Jiang B (2009) Optimization of culture medium for the production of phenyllactic acid by Lactobacillus sp. SK007. Bioresour Technol 100:13661370. doi:10.1016/j.biortech.2008.08.010 Neish AC, Blackwood AC et al (1947) Production and properties of 2, 3-butanediol; dissimilation of glucose by Serratia marcescens. Can J Res 25:6569 Nilegaonkar S, Bhosale SB, Kshirsagar DC, Kapadi AH (1992) Production of 2, 3-butanediol from glucose by Bacillus licheniformis. World J Microbiol Biotechnol 8:378381. doi: 10.1007/BF01198748 Oh IJ, Kim DH, Oh EK, Lee SY, Lee J (2009) Optimization and scale-up of succinic acid production by Mannheimia succiniciproducens LPK7. J Microbiol Biotechnol 19:167171. doi:10. 4014/jmb.0807.447 Parekh M, Formanek J, Blaschek HP (1999) Pilot-scale production of butanol by Clostridium beijerinckii BA101 using a low-cost fermentation medium based on corn steep water. Appl Microbiol Biotechnol 51:152157. doi:10.1007/s002530051375 Perego P, Converti A, Borghi AD, Canepa P (2000) 2, 3-Butanediol production by Enterobacter aerogenes: selection of the optimal conditions and application to food industry residues. Bioprocess Eng 23:613620. doi:10.1007/s004490000210 Plackett RL, Burman JP (1946) The design of optimum multifactorial experiments. Biometrika 33:305325. doi:10.1093/biomet/33. 4.305

respectively. After the optimization, the 2,3-BD concentration increased to 66.5 g/l over a fermentation period of 30 h for the fed-batch culture when using the optimized culture medium and the 2,3-BD productivity reached 2.22 g/l h. It was shown that statistical experimental design offered an effect and feasible approach for 2,3-BD fermentation medium optimization. This newly isolated GRAS strain B. amyloliquefaciens B10-127 showed a higher 2,3-BD production potency than that of K. pneumoniae, which had been demonstrated for its potential on an industrial scale. Furthermore, the optimum medium for 2,3-BD production by this strain mainly composed of inexpensive nutrient sources (common and cheap inorganic salts, a small quantity of corn steep liquor, soybean meal, and ammonium critrate) that are available for efcient and economical production of 2,3-BD on a large scale. Although cell concentration and 2,3-BD concentration were decreased slightly, the scale-up experiment from ask to fermentor showed the possibility of extended application to commercial production processes. So B. amyloliquefaciens B10-127 should be an excellent candidate for the microbial fermentation of 2,3-BD on an industrial scale.
Acknowledgments This work was supported by the Program for New Century Excellent Talents in University (NCET-10-0459), the National Natural Science Foundation of China (30970056), the Hightech Research and Development Programs of China (2007AA 02Z207), the Fundamental Research Funds for the Central Universities (JUSRP31001), the Program of Introducing Talents of Discipline to Universities (111-2-06) and a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions.

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