Research Article pubs.acs.org/journal/ascecg
Production of High Levels of Chirally Pure D‑2,3-Butanediol with a Newly Isolated Bacillus Strain Peng-Fei Yan,†,⊥ Jun Feng,†,⊥ Sheng Dong,† Mei Wang,‡ Ikhlas A. Khan,‡,§ and Yi Wang*,†,∥ †
Department of Biosystems Engineering, Auburn University, 350 Mell Street, Auburn, Alabama 36849, United States The National Center for Natural Products Research, University of Mississippi, 1558 University Circle, University, Mississippi 38677, United States § Division of Pharmacognosy, Department of BioMolecular Sciences, School of Pharmacy, University of Mississippi, University, Mississippi 38677, United States ∥ Center for Bioenergy and Bioproducts, Auburn University, 559 Devall Drive, Auburn, Alabama 36849, United States ‡
S Supporting Information *
ABSTRACT: 2,3-Butanediol (2,3-BD), especially when it exists in the chirally pure form, is a valuable chemical feedstock with numerous applications in various industries. However, in most cases, the 2,3-BD naturally produced by the microorganism is a mixture of two of the three isomers (D-2,3-BD, L-2,3-BD, and meso-2,3-BD), which has restricted applications and thus a much lower value than the chirally pure one. In this study, we report a new isolate Bacillus spp. FJ-4 strain, which can produce highly chirally pure D-2,3-BD (>99.9%) to high levels, with very high productivity and yield. The composition of the fermentation medium for the strain was sequentially optimized using statistical experimental methodologies. During a fed-batch experiment, 100.0 g/L of D-2,3-BD was produced from 226.8 g/L glucose, with a yield of 0.44 g/g (88.2% of the theoretical maximum). Acetoin was the primary byproduct, and no other byproduct such as lactate, acetate, or ethanol was detected, which would be advantageous for the downstream purification process. Moreover, no special conditions such as limited oxygen level (which generally limits the cell growth and thus final D-2,3-BD production) are required for the fermentation. These results indicated that Bacillus spp. FJ-4 is a robust workhorse for efficient D-2,3-BD production from low-value carbon sources. KEYWORDS: 2,3-Butanediol, Chirally pure 2,3-butanediol, D-2,3-Butanediol, Bacillus, Medium optimization, Response surface methodology (RSM), Fed-batch
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INTRODUCTION 2,3-Butanediol (2,3-BD) is an important platform chemical that has applications in pharmaceutical, food, cosmetic, and plastic industries.1−3 In general, 2,3-BD exists in three stereoisomeric forms: D-2,3-BD, L-2,3-BD, and meso-2,3-BD. Each isomer is a valuable compound and has its own unique applications. For example, L-2,3-BD and meso-2,3-BD could be used as building blocks in the synthesis of chiral compounds,2,4 while enantiomerically pure D-2,3-BD can be employed not only as the precursor for producing various chiral compounds or intermediates, but also as an antifreeze agent thanks to its low freezing point.3 Currently, 2,3-BD can be produced by either the petroleum-based chemical synthesis approach or the microbial fermentation-based biological approach.5 The chemical synthesis of 2,3-BD from petroleum is highly energyintensive and meanwhile generates various secondary pollutants.4,6 Comparatively, the microbial production of 2,3-BD is easier to achieve, less expensive, and more environmentally friendly, thus attracting a lot of interest in recent years. © XXXX American Chemical Society
Although there are a lot of microorganisms capable of producing 2,3-BD, many of the prominent 2,3-BD producing strains, such as Klebsiella oxytoca, K. pneumonia, and Serratia marcescens, are pathogenic.5,7,8 Thus, there are serious safety concerns for the production of 2,3-BD using these microorganisms on the industrial-scale.9 On the other hand, there are also nonpathogenic species including Paenibacillus polymyxa, Bacillus licheniformis, and B. amyloliquefaciens, that can produce high levels of 2,3-BD.10−12 However, almost all the wild-type microorganisms mentioned above (either pathogenic or nonpathogenic) produce a mixture of two of three isomers of 2,3-BD, with the ratio depending on the strain and the fermentation condition.5 Due to the restricted applications, the mixed 2,3-BD has a much lower value than the chirally pure one, while in order to purify a target form of 2,3-BD Received: August 21, 2017 Revised: September 7, 2017
A
DOI: 10.1021/acssuschemeng.7b02910 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX
Research Article
ACS Sustainable Chemistry & Engineering
Table 1. Variables To Be Screened in the Plackett−Burman Design Experiment and the Statistical Analysis of the Plackett−Burman Design Experiment Resultsa factor
variable
low level
high level
coeff
std error
T-value
P-value
glucose(g/L) tryptone−yeast extract mixturec (g/L) (NH4)2SO4 (g/L) MgSO4 (g/L) trace element solution (μM/L)d PBS (mM/L)
X1 X2 X3 X4 X5 X6
80 9 2 0.4 2 15
100 15 4 0.8 4 30
6.16 2.96 −0.56 −1.68 0.21 −0.097
0.36 0.36 0.36 0.36 0.36 0.36
17.082 8.197 −1.552 −4.657 0.582 −0.268
99%).14 However, as these reported strains need to be grown under low oxygen conditions, the cell density and the overall 13,14 D-2,3-BD productivity were generally low. B. subtilis, which is generally recognized as safe (GRAS) by the US Food and Drug Administration,15 is naturally distributed in various environments. Numerous B. subtilis strains have been isolated and employed for the production of various valuable compounds.15,16 In this study, we report a new Bacillus spp. FJ-4 strain, which we recently isolated; it can produce highly enantiomerically pure D-2,3-BD to high levels, with very high productivity and yield. We first optimized the composition of the fermentation medium for the strain using statistical experimental methodologies. Furthermore, we carried out fed-batch fermentation using the optimized medium for efficient D-2,3-BD production. Our results demonstrated that Bacillus spp. FJ-4 is a robust workhorse for D-2,3-BD production from low-value carbon sources.
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Y = β0 +
∑ βi Xi
i = 1, 2, ..., k
(1)
where Y was the predicted response (D-2,3-BD production), β0 was the model intercept, βi was the linear coefficient, and Xi was the coded levels of the independent factor. Each variable had two levels: high level (+1) and low level (−1) (Tables 1 and 2). In this work,
Table 2. Plackett−Burman Design and the Corresponding D-2,3-BD Production under Each Condition variable levels
EXPERIMENTAL SECTION
Chemicals. All of the chemicals used in this study, including the ones for strain cultivation and fermentation, were purchased either from VWR (Radnor, PA) or Sigma-Aldrich (St. Louis, MO). D-2,3-BD (98.0%), L-2,3-BD (99.0%), and meso-2,3-BD (99.0%) were purchased from Sigma-Aldrich (St. Louis, MO). Strain Isolation. To isolate the Bacillus strain, specimens (∼1 g of each) were taken from various fermented foods (Chinese natto and sufu from the local grocery store), suspended in 100 mL of phosphatebuffer saline (PBS) solution, and then treated at 80 °C for 5 min. After centrifugation, the supernatant was collected, which was then diluted in series and spread onto Luria−Bertani (LB) plates. The plates were incubated at 37 °C for 20 h. Single colonies from the plates were picked and restreaked for three times in order to obtain the pure culture. Afterward, fermentations were carried out at 37 °C using the isolates with the medium containing (in g/L) glucose 100, yeast extract 10, tryptone 3, (NH4)2SO4 2, and MgSO4 0.6. After 96 h of fermentation, samples were taken, and the concentration of 2,3-BD was measured. Strains that produced the highest 2,3-BD were selected for further characterization. Microorganism and Cultivation Methods. The Bacillus spp. FJ-4 strain for D-2,3-BD production used in this work was stored in 30% glycerol at −80 °C. For the preparation of seed culture for fermentation, the glycerol stock of Bacillus spp. FJ-4 was revived by growing it on agar slants containing LB medium. Single colonies were then picked and inoculated into 5 mL of LB liquid medium, and cultivated on a rotary shaker (200 rpm) for 10 h. Seed culture was
run
X1
X2
X3
X4
X5
X6
D-2,3-BD
1 2 3 4 5 6 7 8 9 10 11 12
1 −1 1 −1 −1 −1 1 1 1 −1 1 −1
1 1 −1 1 −1 −1 −1 1 1 1 −1 −1
−1 1 1 −1 1 −1 −1 −1 1 1 1 −1
1 −1 1 1 −1 1 −1 −1 −1 1 1 −1
1 1 −1 1 1 −1 1 −1 −1 −1 1 −1
1 1 1 −1 1 1 −1 1 −1 −1 −1 −1
47.9 36.7 38.1 34.3 31.9 29.5 44.1 50.5 49.9 33.6 41.6 32.2
± ± ± ± ± ± ± ± ± ± ± ±
(g/L) 0.1 0.0 0.6 0.7 0.3 0.5 0.3 0.2 1.0 0.5 0.2 0.1
the symbol code and actual levels of each variable employed in the experimental design are illustrated in Table 1, while the detail of the Plackett−Burman design was shown in Table 2. There were 12 experiments performed to evaluate the effects of six variables on D-2,3-BD production in the fermentation with Bacillus spp. FJ-4. The D-2,3-BD production in each experiment was taken as the response. Path of Steepest Ascent. The path of steepest ascent is a method for moving the response sequentially to the optimal region in the direction of maximum response increase.18 On the basis of results from the experiments following the Plackett−Burman design, a path of steepest ascent was determined. Then, a series of experiments was carried out along the path (toward the maximum of the response; Table 3) until no additional increase in response was evident. B
DOI: 10.1021/acssuschemeng.7b02910 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX
Research Article
ACS Sustainable Chemistry & Engineering
3-D response surface plotting. All of the optimization experiments were conducted in 250 mL flasks containing 100 mL of medium with agitation at 150 rpm at 37 °C. Samples were collected at 120 h for each fermentation, and the D-2,3-BD concentration was measured. Fed-Batch Fermentation. Fed-batch fermentation was conducted in a BioFlo 115 benchtop fermenter (New Brunswick Scientific Co., Enfield, CT) with a working volume of 1.5 L. The fermentation was performed at 37 °C, with agitation of 300 rpm and an air flow rate of 2.0 vvm. The pH was maintained at 6.5 by automatic addition of 5 M NaOH or 5 M HCl programmed by the NBS BioCommand software (New Brunswick Scientific Co.). Samples were collected over the fermentation process to determine the cell growth, and concentrations of glucose, D-2,3-BD, and other products. Analytical Methods. Cell growth was determined by measuring the optical density at 600 nm (OD600) with an Ultrospec 10 cell density meter (Amersham Biosciences Corp., Piscataway, NJ). The concentration of glucose and the metabolites including D-2,3-BD, acetoin, ethanol, lactate, and acetate was measured using a highperformance liquid chromatography instrument (HPLC, Agilent Technologies 1260 Infinity series, CA), equipped with a Bio-Rad Aminex HPX-87H column (300 mm × 7.8 mm) which was operated at 65 °C. The mobile phase used was 0.005 M sulfuric acid with a flow rate of 0.5 mL/min. The stereoisomers of 2,3-BD in the broth were analyzed using an Agilent 7890B gas chromatograph equipped with an Agilent 5975C mass spectrometer. The chromatographic separation was achieved with an Agilent J&W CYCLOSIL-B column (30 m × 0.25 mm, 0.25 μm). Helium was used as the carrier gas with a flow rate of 1.5 mL/min. The injection volume was 0.2 μL with a split ratio of 25:1. The oven temperature was maintained at 50 °C for 2 min, then increased at a rate of 3 °C/min to 100 °C, and further increased at a rate of 30 °C/min to 200 °C. Mass spectra were obtained in the electron ionization mode (70 eV). Acquisition was started after an 8 min delay using the scan mode with m/z from 40 to 400.
Table 3. Experimental Design of the Path of Steepest Ascent and the Corresponding D-2,3-BD Production run
glucose (g/L)
tryptone−yeast extract mixture (g/L)
MgSO4 (g/L)
1 (initial) 2 3 4 5
90 110 130 150 170
12 15 18 21 24
0.6 0.5 0.4 0.3 0.2
D-2,3-BD
(g/L) 37.8 48.6 40.9 40.5 38.3
± ± ± ± ±
0.0 0.4 1.0 1.0 1.2
Therefore, the method of steepest ascent helped determine the levels of variables close to the optimal, which could then be used as the center point for the central composite design (CCD) as detailed below. Central Composite Design (CCD) and Response Surface Methodology (RSM). Response surface methodology (RSM) based on central composite design (CCD) was employed to optimize the most significant factors screened by the Plackett−Burman design for improved D-2,3-BD production. Three variables (X1, X2, and X4) with five levels (−1.682, −1, 0, +1, +1.682) were investigated, and a set of 20 experiments were performed (Table 4). The variables were coded according to the following equation: Xi =
(xi − x0) Δxi
i = 1, 2, ..., k
(2)
where Xi was the dimensionless coded value of the independent variable, xi was the actual value of the independent variable, x0 was the actual value of the independent variable at the center point, and Δxi was the step change. The statistical analysis to predict the optimal level of variables was explained by the following quadratic equation:
Y = β0 +
∑ βi Xi + ∑ βiiXi2+ ∑ βijXiXj
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i , j = 1, 2, ..., k
(3) where Y was the predicted response (D-2,3-BD production), β0 was the intercept coefficient, βi was the linear coefficient, βii was the squared coefficient, βij was the interaction coefficient, and Xi and Xj were the coded independent variables. The “Design Expert” software package (Version 8.0.7.1, Stat-Ease Inc., Minneapolis, MN) was used for all the statistical analysis and the
RESULTS AND DISCUSSION Strain Isolation for D-2,3-BD Production. In total, 10 isolates were obtained from the fermented food. Among them, strain FJ-4 produced the highest concentration of 2,3-BD, and thus was selected for further investigation. The 16S rDNA gene of strain FJ-4 was amplified by PCR and sequenced. On the
Table 4. Central Composite Design (CCD) and the Corresponding D-2,3-BD Production coded variable levels
real variable levels
D-2,3-BD
(g/L)
run
X1
X2
X4
glucose (g/L)
tryptone−yeast extract mixture (g/L)
MgSO4 (g/L)
obsd
predicted
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 −1.682 1.682 0 0 0 0 0 0 0 0 0 0
−1 −1 1 1 −1 −1 1 1 0 0 −1.682 1.682 0 0 0 0 0 0 0 0
−1 −1 −1 −1 1 1 1 1 0 0 0 0 −1.682 1.682 0 0 0 0 0 0
90 130 90 130 90 130 90 130 76.36 143.64 110 110 110 110 110 110 110 110 110 110
12 12 18 18 12 12 18 18 15 15 9.954 20.046 15 15 15 15 15 15 15 15
0.4 0.4 0.4 0.4 0.6 0.6 0.6 0.6 0.5 0.5 0.5 0.5 0.332 0.668 0.5 0.5 0.5 0.5 0.5 0.5
41.0 43.4 43.3 44.3 39.6 40.9 41.6 42.8 35.0 41.6 44.1 49.8 43.5 42.7 47.2 47.5 47.5 47.5 47.8 47.4
40.2 43.3 42.8 45.2 39.0 41.6 42.0 43.9 36.0 40.1 44.7 48.8 43.9 41.8 47.5 47.5 47.5 47.5 47.5 47.5
C
DOI: 10.1021/acssuschemeng.7b02910 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX
Research Article
ACS Sustainable Chemistry & Engineering
Figure 1. GC−MS chromatograph profiles of D- and/or L-2,3-BD: (A) the mixture of standard samples of D- and L-2,3-BD, (B) the standard sample of L-2,3-BD, (C) the standard sample of D-2,3-BD, (D, E) 2,3-BD produced from the fermentation with Bacillus spp. FJ-4 (samples from two independent fermentation runs were tested, respectively).
sources, MgSO4, inorganic salts (containing equal molar concentrations of FeSO4·7H2O, CaCl2, ZnCl2, MnSO4·H2O, NiCl2· 6H2O, and CoCl2·6H2O), and PBS (composed of KH2PO4 (68.5%) and K2HPO4 (31.5%) to obtain a pH at ∼6.5) were investigated through single factor experiments. Results suggested that levels of glucose, organic nitrogen source (tryptone−yeast extract, or TP-YE mixture), (NH4)2SO4, MgSO4, trace elements solution, and PBS were all major variables influencing D-2,3-BD production (data not shown). Therefore, these factors (along with the approximate range of levels determined by preliminary experiments) were selected for further optimization through experiments with the Plackett−Burman design (Table 1). Table 2 showed the details of a 12-run Plackett−Burman experimental design along with the corresponding D-2,3-BD production. The effect of each variable on the response and the significant level were summarized in Table 1. In order to approach the neighborhood of the optimal response for D-2,3-BD production, the fitted first-order model
basis of the sequence results, strain FJ-4 was identified as a strain of Bacillus spp. (data not shown). As shown in Figure S1, the HPLC measurement result indicated that there was only D-2,3-BD and/or L-2,3-BD, but no meso-2,3-BD produced in the fermentation with strain FJ-4. To further identify the stereoisomeric form of the 2,3-BD produced by the FJ-4 strain, a GC−MS system equipped with a β-cyclodextrin-based column for chiral separations was employed. As shown in Figure 1, standard samples of D-2,3-BD and L-2,3-BD can be well-separated using the GC−MS, and results confirmed that the FJ-4 strain produced chirally pure D-2,3-BD (with a purity of >99.9%). Optimization of Fermentation Medium for 2,3-BD Production with Plackett−Burman Design. Preliminary experiments were first conducted to determine the important factors of the fermentation medium that affected the 2,3-BD production in FJ-4 strain. Factors include the initial glucose concentration, organic nitrogen source, inorganic nitrogen D
DOI: 10.1021/acssuschemeng.7b02910 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX
Research Article
ACS Sustainable Chemistry & Engineering
analysis, a second-order polynomial model was obtained as follows:
was acquired on the basis of the results from the Plackett− Burman design experiments:
Y = 47.50 + 1.23X1 + 1.22X 2 − 0.62X4 − 3.34X12
Y = 39.18 + 6.16X1 + 2.96X 2 − 0.56X3 − 1.68X4 + 0.21X5 − 0.097X6
− 0.27X 22 − 1.64X42 − 0.18X1X 2 − 0.13X1X4
(4)
+ 0.099X 2X4
The coefficient of each factor in eq 4 represented the extent of its effect on the D-2,3-BD production. The linear regression coefficient (R2) of the polynomial model equaled 0.987, suggesting that 98.7% of the variability in responses can be explained by the model. The adjusted determination coefficient was also very high (Adj R2 = 0.972), indicating the model was well-suited for simulating the Plackett−Burman design. Statistical analysis results showed that X1 (glucose; p < 0.0001), X2 (TP-YE mixture; p = 0.0004), and X4 (MgSO4; p = 0.0055) were the factors exhibiting the most significant impacts on the D-2,3-BD production (Table 1). Specifically, X1 and X2 had positive effects within this tested range which were indicated by the positive coefficients, while X4 had negative effects (with a negative coefficient). In the fermentation, glucose and the TP-YE mixture act as the primary carbon source and nitrogen source, respectively. On the other hand, Mg2+ plays an important role in the conversion of pyruvate to acetoin and 2,3-BD, because the activity of the key enzyme (α-acetolactate synthase) in this process is Mg2+-dependent.19 These factors (X1, X2, and X4) were selected for further optimization as detailed below using “the path of steepest ascent”. During such optimization, other factors including X3 ((NH4)2SO4) and X6 (PBS) were set at their low levels due to the negative effects, and X5 was set as its high level due to the positive effect (Table 1). Further Optimization with the Path of Steepest Ascent. On the basis of the above results, further optimization with the path of steepest ascent was applied to obtain factor levels even closer to the optimal response scope, i.e., increased concentration of glucose and TP-YE mixture (since they had positive effects on the response within the tested range, Table 1), while the MgSO4 concentration decreased (since it had negative effect on the response within the tested range, Table 1). The zero level (glucose, 90 g/L; TP-YE mixture, 12 g/L; MgSO4, 0.6 g/L) from the Plackett−Burman design experiment was set as the base point of the path. The coefficients of X1, X2, and X4 (+6.16, +2.96, and −1.68, respectively) were approximately proportional to (+4.0, +2.0, −1.0), determining the step size to be used for the path of steepest ascent. The design and corresponding responses of the steepest ascent were illustrated in Table 3. The D-2,3-BD production was the highest when the concentration of glucose, TP-YE mixture, and MgSO4 were at 110, 15, 0.5 g/L, respectively, which indicated that these levels of the three variables gave the fermentation condition leading to the D-2,3-BD production very close to the region of optimum D2,3-BD response. Central Composite Design (CCD) and Response Surface Methodology (RSM). To further obtain the optimal level of glucose, TP-YE mixture, and MgSO4 for D-2,3BD production, CCD and RSM were employed. The experimental design, together with the responses of CCD, was shown in Table 4. The levels of the three variables obtained from the path of steepest ascent experiment were used as the center point (zero level for the CCD). Through the fitting of the observed data by applying the multiple regression
(5)
where Y was the predicted D-2,3-BD production (g/L), and X1, X2, and X4 were the coded values of glucose, TP-YE mixture, and MgSO4, respectively. The significance of the fitted model was tested by ANOVA. The F-value and P-value were 27.36 and 0.0001, respectively. The regression coefficient R2 of 0.961 for this model indicated that 96.1% of the sample variation for the 2,3-BD production can be explained by the model.20 The value of Adj R2 was also very high (0.926). All of these results indicated that the model possessed a high significance, and it was very suitable for use in simulations of the CCD experiment. The P- and T-values were usually used to evaluate the significance of each variable, and they could also reflect the strength of interactions between each independent variable.21 The larger T-value as well as the smaller P-value indicate that the corresponding variable is more significant.20 As shown in Table 5, Table 5. Statistics for the Regression of the Optimization Model Using the Response Surface Methodology (RSM)a statistical analysis variable
coeff
std error
T-value
P-value
intercept X1 X2 X4 X21 X22 X24 X1X2 X1X4 X2X4
47.50 1.23 1.22 −0.62 −3.34 −0.27 −1.64 −0.18 −0.13 0.099
0.40 0.26 0.26 0.26 0.26 0.26 0.26 0.34 0.34 0.34
119.85 4.69 4.63 −2.36 −13.03 −1.07 −6.41 −0.53 −0.37 0.29
