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Optimization of process parameters for poly γ-glutamate production under solid state fermentation from Bacillus subtilis CCTCC202048
Process Biochemistry 40 (2005) 3075–3081 www.elsevier.com/locate/procbio
Optimization of process parameters for poly g-glutamate production under solid state fermentation from Bacillus subtilis CCTCC202048 Xu Jian a,c, Chen Shouwen a,b,c,*, Yu Ziniu a,b,c a National Key Laboratory of Agricultural Microbiology, Wuhan 430070, PR China National Engineering Research Center of Microbial Pesticides, Wuhan 430070, PR China c Department of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, PR China b
Received 20 October 2004; received in revised form 2 February 2005; accepted 14 March 2005
Abstract The solid state fermentation (SSF) was carried out for the production of poly g-glutamate (PGA) from a new strain Bacillus subtilis CCTCC202048. The fermentation factors, which included composition of substrates, initial moisture content, incubation temperature, fermentation period, and additional nutrients such as mineral salts, carbon sources and nitrogen sources, were investigated and optimized. The maximum PGA production (83.61 g/kg of dry substrate) was obtained in the mixed substrates of soybean cake powder and wheat bran (11:9, w/w) supplemented with glutamate (40.14 g/kg), citric acid (18.50 g/kg), NH4NO3 (20.05 g/kg) and mineral salts (MgSO47H2O, CaCl22H2O, FeCl36H2O and MnSO4H2O), with initial moisture content as 65%, at 40 8C incubated for 42 h. This yield was four times more than that before optimization. This is the first report on optimization of PGA production in SSF. # 2005 Elsevier Ltd. All rights reserved. Keywords: Poly g-glutamate (PGA); Solid state fermentation; Bacillus subtilis; Optimization; Response surface experiment; Canonical analysis
1. Introduction Poly g-glutamate (PGA) is an unusual anionic, watersoluble and high viscous polypeptide in which glutamate is polymerized via g-amide linkage. It was first discovered in the capsule of B. anthracis and later found in the cell surrounding of other nonpathogenic Bacillus spp. including B. licheniformis, B. megaterium, Bacillus subtilis, and B. amyloliquefaciens [1]. For its excellent characters such as high efficient water-absorption, completely biodegradable and non-toxic to human, PGA has attracted a variety of interest in the past decade and proved to be a safe and environmental friendly material that has potential applications in a wide range of industry and agriculture such as food, cosmetics, medicine and manure [2,3]. PGA and its derivatives can be used as thickener, humectant, drug carrier, heavy metal absorber, feed additives [4–8], etc. Even though the strategies for the production of PGA in submerged fermentation (SmF) have been researched by several groups * Corresponding author. Tel.: +86 27 87286972; fax: +86 27 87393882. E-mail address: [email protected] (C. Shouwen). 1359-5113/$ – see front matter # 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2005.03.011
[9–11], the fermentation technology for PGA still remains difficult in respect that the significant increase in viscosity of the media results in uncontrollable foaming, limitation of the volumetric oxygen mass transfer which leads to insufficient cell growth and a decrease in PGA yield [12]. Furthermore, a relative high expenditure for SmF media hampers PGA becoming a prevalent commercial valuable product. Solid state fermentation (SSF) is an important mode of fermentation that microorganisms grow on or within the substrates or supports, in the absence or near-absence of free water and excrete aimed products efficiently [13,14]. Considering the lower energy requirement, simplicity of cultivation equipment and high product yield, this traditional method ignites the interest of researchers again recently and has been widely employed in the production of enzymes, fine chemicals, antibiotics [15–17], etc. PGA can be produced by microorganism under SSF since there is a Japanese traditional fermented food Natto made from soybeans by B. subtilis possessing of viscous materials which were mainly composed of PGA. SSF offers an alternative to solve many troubles encountered in SmF for the production of PGA. Firstly, the uncontrollable foaming
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in SmF does not appear in SSF, which will relieve the complexity of controls demanded in SmF to a great extent. Secondly, the expenditure for the media and energy is going to be reduced largely because the cheaper substrates usually composed of agro-industry byproducts or residuals under SSF are used and static cultivation is achieved. Reports on PGA production by SSF are lacking in the literature. In the present study, the aim was to investigate the production of PGA in SSF and achieve a series of optimal process parameters. A newly isolated strain B. subtilis CCTCC202048 that could produce PGA in SmF was employed in this study. The factors considered critical to PGA production in SSF were studied and optimized in this paper.
2. Materials and methods 2.1. Microorganisms and inoculum preparation A new strain of B. subtilis CCTCC202048, isolated from soil samples and developed in this laboratory, was used in the present study. It was maintained on LB agar (tryptone, 10 g/l; yeast extract, 5 g/l; NaCl, 10 g/l; Agar 15 g/l; pH 7.0), stored at 4 8C, and subcultured every 4 weeks. A loopful of the stored strain was transferred to 50 ml LB liquid medium (tryptone, 10 g/l; yeast extract, 5 g/l; NaCl, 10 g/l; pH 7.0), which was incubated in 250 ml flask at 200 rpm at 37 8C. When the turbidity of the culture reached about 4.0 at 600 nm (6 109 cells/ml), cells were harvested by centrifugation at 8000 rpm for 10 min, washed with 0.85% NaCl solution, and then suspended in 50 ml of 0.85% NaCl solution. 2.2. Optimization studies for PGA production in SSF
to the bran was 1:1 (w/w) and the temperature of fermentation was kept at 37 8C. Initial moisture of substrates was adjusted to 50%. The yield of PGA was determined at 36 h. After choosing the best powder substrate, the proportion (8:12, 9:11, 10:10, 11:9, 12:8, 13:7, w/w) between the chosen powder and the wheat bran was optimized too. 2.2.2. Effect of initial moisture content The moisture content of the substrates was adjusted by adding distilled water before autoclaving. The effect of initial moisture content (50%, 55%, 60%, 65%, 70%, w/w) was investigated. The other process parameters were kept identically in this study. 2.2.3. Effect of incubation temperature and period The yields of PGA in six batches fermentation conducted at different temperature (30 8C, 33 8C, 37 8C, 40 8C, 43 8C) were investigated from 6 h to 72 h at an interval of 6 h. The other conditions were kept at their optimized level. 2.2.4. Effect of additional mineral salts Different mineral salts such as MgSO47H2O, CaCl22H2O, FeCl36H2O and MnSO4H2O were dissolved separately in distilled water used to adjust the moisture of the solid substrates. According to previous studies on the effect of salts on PGA production in SmF by B. subtilis [18], the final concentrations for each additional salt in solid media were set to be 0.5 g/kg, 0.15 g/kg, 0.04 g/kg and 0.104 g/kg, respectively, by the order mentioned above. The yield of PGA was determined to study their effect on the final product. 2.2.5. Effect of additional carbon sources Different carbon sources: glucose, lactose, sucrose, starch, citric acid, glycerol and glutamate were added into the solid substrate separately to a final concentration of 20 g/ kg for testing their effect on the yield of PGA.
The static experiments were conducted in 250 ml flasks containing 20 g (dry weight) solid substrates and proper volume of distilled water or nutritious solution, which were sterilized at 121 8C for 30 min prior to fermentation. The initial pH was adjusted to 7.0 with NaOH and inoculum level was 5%. The parameters optimized were as follows: selection of solid substrate and their ratio, initial moisture content, temperature, incubation time, additional carbon sources, additional nitrogen sources and salts additives. The strategy adopted for optimization of the above parameters was consecutive evaluation. Initially one parameter was evaluated and then it was incorporated at its optimized level in the subsequent experiments. The unit ‘g/kg’ quoted in this paper denotes theweight of component per kilogram of dry substrate.
2.2.6. Effect of additional nitrogen sources Different nitrogen sources: NH4Cl, NaNO3, NH4NO3, peptone and yeast extract were added to the solid substrate separately for testing their effect on the yield of PGA. A consistent amount of additive elemental nitrogen (5 g/kg) was needed in this experiment, because the consumption efficient of different type of nitrogen sources would be identified by doing so. The concentration of each additional supplementary is shown in Table 2 according to the content of elemental nitrogen in different nitrogen sources. And when this experiment was conducted, solid substrates were not supplemented with additional carbon sources.
2.2.1. Effect of different solid substrates and their ratio Different effects of four kinds of substrates (the powder of soybean cake, rapeseed cake, cottonseed cake and peanut cake were separately mixed with wheat bran) on PGA production were studied. The proportion of the cake powder
2.2.7. Combination of optimized components Effects of optimized components on PGA production were studied by supplementing combinations of the additional carbon sources components and additional nitrogen sources components chosen in the foregoing two
X. Jian et al. / Process Biochemistry 40 (2005) 3075–3081
experiments. The response surface experiment was conducted for optimizing the concentration of each component using Box-Behnken design in this step. SAS, version 8.01 (Institute Inc., Cary, NC, USA) was used for the experimental design and regression analysis of the experimental data obtained. Canonical analysis was one part of the process RSREG output.
Table 1 Effect of different substrates on PGA production Substrate mixed with wheat brana
PGA production (g/kg)b
Soybean cake powder Rapeseed cake powder Cottonseed cake powder Peanut cake powder
17.89 8.73 7.94 13.45
a b
2.3. PGA extraction
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The ratio of wheat bran and other powder is 1:1. g/kg indicates g PGA/kg dry substrate.
A weighted quantity of fresh fermented substrates were treated in distilled water (1:10, w/v) in flasks and mixed thoroughly on a shaker at 200 rpm for 1 h. The mixtures were filtered through two-layer muslin cloth and the obtained extracts were centrifuged at 12,000 rpm for 15 min. Clarified supernatants were collected for PGA determination. 2.4. PGA determination PGA concentration was detected using the method of Do et al. [19] by HPLC. The quantification system equipped with Waters 515 HPLC pump, Waters 2487 dual l absorbance detector (Milford, MA), and TSK-GEL G6000PWXL column (Tosho, Tokyo, Japan). A mixture of 25 mM Na2SO4 solution-acetronitrile (4:1, v/v) was used as a mobile phase at a flow rate of 0.6 ml/min.
3. Results and discussion 3.1. Optimization of different solid substrates and their ratio The selection of a suitable substrate is a critical factor for a SSF process. There were three points as following needed to be considered: (1) Substrate is not only nutrient material for growth of microorganisms, but also the supporter for microorganisms, giving enough room for oxygen transfer and heat dispersion. (2) In prior research, it was observed that high protein content material was a perfect substrate for B. subtilis CCTCC202048 producing PGA. (3) Wheat bran, one part of carbon sources, can be a satisfying supporter and carrier because of its porosity and cheapness. According to these points above, the initial composition of substrate was designed like this: making the high protein content material powdery, for the sake of increasing substrate consuming rate, and mixing the powder with wheat bran in proper proportion. In the four tested high protein content agro-industrial residues, the powder of soybean cake media proved superior to the other media. The results are shown in Table 1. The ratio between soybean cake powder and wheat bran is an important factor, because overabundance of powder would decrease the porosity of media. To select an optimized ratio, seven kinds of combination were investigated. The highest PGA production was 19.30 g/kg of dry substrate as the results shown (Fig. 1),
Fig. 1. Effect of substrates ratio on PGA production (WP, weight of soybean cake power; WB, weight of wheat bran).
while the ratio between soybean cake powder and wheat bran was 11:9 (w/w). 3.2. Optimization of initial moisture content The initial moisture level is one of the most important factors in SSF media. Five moisture levels ranging from 50% to 70% were established to study their effect on PGA production and the results obtained are shown in Fig. 2. Significantly, the highest production was attained when the initial moisture levels were 60%–65%. Either low or high initial moisture would decrease the final product for the reason that low moisture substrates reduce mass transfer process and high moisture substrates reduce the porosity of the wheat bran [17]. 3.3. Optimization of incubation temperature and period On accounting of mutual interaction affecting the production of PGA between the fermentation temperature and period, the concentrations of PGA in solid media have been determined at an interval of 6 h from 6 h to 72 h at different incubation temperature conditions. Results are shown in Fig. 3. The highest production of PGA was obtained at 40 8C after 42 h. Despite high temperature the moisture content of medium was maintained well during fermentation. It was 54% at 42 h while the initial moisture
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Fig. 2. Effect of initial moisture content on PGA production.
content was 60%. The reason was that enough water was kept by PGA, which had the character of high waterabsorbance [5]. In addition, the end-point of fermentation should be carefully controlled because synthesized PGA would be biodegraded by depolymerase that reported by King et al. [20] at the later stationary phase. At the fermentation temperature of 40 8C, the maximum concentration of PGA in media reached 39.50 g/kg at 42 h and decreased sharply after 48 h. 3.4. Optimization of single additional nutrients The strain produced more PGA caused by each additional salt compared to the production without additional salt as the results shown in Fig. 4. These salts may have the positive effect on enzyme reaction as the composition of co-enzyme in the metabolism of the strain, thus they increased the yield of PGA [2,18,21].
Fig. 3. Effect of incubation temperature and period on PGA production.
Fig. 4. Effect of supplementary mineral salts on PGA production (1, MgSO47H2O; 2, CaCl22H2O; 3, FeCl36H2O; 4, MnSO4H2O; 5, control).
In the seven kinds of carbon sources which were added into solid substrates, glutamate and citric acid enhanced the PGA production by 31.0% and 20.0%, which reached 59.20 g/kg and 54.21 g/kg, respectively. The results are shown in Fig. 5. It was deduced that additional glutamate was material for synthesizing PGA as Ogawa reported in B. subtilis MR-141 [22]. Additional citric acid could be transferred to a-ketoglutaric by the TCA cycle and the aketoglutaric was then converted to glutamate through the glutamate synthetic pathway. This part glutamate was also an important source for consisting PGA unit as described by Goto and Kunioca in B. subtilis IFO3335 [23]. According to those reports and results, it was presumed that the glutamate units of PGA derived from both additional glutamate and the
Fig. 5. Effect of supplementary carbon sources on PGA production (1, glucose; 2, lactose; 3, sucrose; 4, starch; 5, citric acid; 6, glycerol; 7, glutamate; 8, control).
X. Jian et al. / Process Biochemistry 40 (2005) 3075–3081 Table 2 Effect of supplementary nitrogen sources on PGA production
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3.5. Optimization of multiple additional nutrients
Supplementary nitrogen sources
Supplementary concentration (g/kg)a
PGA production (g/kg)
NH4Cl NaNO3 NH4NO3 Peptone Yeast extract Control
19.11 30.36 14.29 41.67 28.57 0
53.56 55.81 59.96 50.23 49.30 44.48
a The Supplementary concentration of elemental nitrogen was unified to 5 g/kg according to corresponding concentration in different nitrogen sources.
glutamate synthesized by glutamate synthetic pathway in this strain. In contrast, glucose, lactose, sucrose and starch were not considered as ideal additional nutrients for that they reduced the PGA production to a great extent. Three kinds of inorganic nitrogen sources (NH4Cl, NaNO3 and NH4NO3) and two kinds of organic nitrogen sources (peptone and yeast extract) were investigated about their effect on PGA production while the additional carbon sources were absent. As the results shown (Table 2), all the nitrogen sources could promote the final product, but the inorganic nitrogen sources were more efficient. The maximum yield of PGA (59.69 g/kg) was obtained by additional NH4NO3 in results. NO3 was observed enhancing the production of PGA more efficiently than NH4+ in this strain in comparing the effect of additional NH4Cl and NaNO3. This character has not been reported in earlier research on PGA production. Kunioka reported that (NH4)2SO4 was assimilated by B. subtilis IFO3335 as a substrate to produce glutamate, which then was converted into PGA [24]. So it was presumed that B. subtilis CCTCC202048 could utilize the additional NO3 and NH4+ as substrates to provide ammonia for synthesizing glutamate and then give a positive effect on PGA production.
Glutamate, citric acid and NH4NO3 were selected as the optimal additional carbon sources and nitrogen sources in the foregoing experiments. Because these kinds of nutrients had shown great efficiency to enhance the PGA production, their additive amounts were worthy to be confirmed. Considering that these three components may have mutual effects on PGA production, they were combined and investigated using Box-Behnken design whose results could be fitted with a second-order polynomial equation by a multiple regression analysis and locate the optimum concentrations in a rapid way. Four kinds of mineral salts were all added to medium, but were not included as factors in this statistical experiment. The reasons were that salts showed insignificant mutual effects with additional glutamate, citric acid and NH4NO3 and their optimum concentration was coincide with the original ones given in the single nutritious experiments (these data are not shown). The experimental design is shown in Table 3, together with the observed data and the predicted values from the model equation. The second-order polynomial equation obtained for PGA production is shown in Eq. (1).
Table 4 ANOVA table of Box-Behnken design experiments Factor
d.f.
SS
R2
F-value
Total model Linear Quadratic Cross product
9 3 3 3
781.08 11.14 683.57 86.37
0.9977 0.0142 0.8731 0.1103
240.74* 10.30* 632.05* 79.86*
Residual Lack of fit Pure error
3 2
1.66 0.14
Total SS
5
1.80
*
7.89
Significant at 5% level.
Table 3 Experimental design and results of the Box-Behnken design together with predicted yield from the model equation Run number
Citric acid (g/kg)
Glutamate (g/kg)
NH4NO3 (g/kg)
Observed PGA yield (g/kg)
Predicted PGA yield (g/kg)
1 2 3 4 5 6 7 8 9 10 11 12 13a 14a 15a
12 12 24 24 12 12 24 24 18 18 18 18 18 18 18
30 50 30 50 40 40 40 40 30 30 50 50 40 40 40
20 20 20 20 10 30 10 30 10 30 10 30 20 20 20
58.82 65.79 66.10 62.91 62.38 70.28 71.61 64.61 62.19 60.93 60.14 63.38 80.42 80.15 79.89
61.89 62.93 63.88 64.92 65.87 66.59 67.86 66.57 60.78 61.50 61.82 62.54 80.15 80.15 80.15
a
Center point.
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Fig. 6. 3D plots of response surface optimization experiment results: (A) the effect of supplementary citric acid concentration, glutamate concentration and their mutual interaction on PGA production. The other factor was held at central point; (B) the effect of supplementary glutamate concentration, NH4NO3 concentration and their mutual interaction on PGA production. The other factor was held at central point.
Table 5 Factor static statistical analysis from the model equation
4. Conclusions
Factor
d.f.
SS
MS
F-value
Citric acid Glutamate NH4NO3
4 4 4
204.77 492.43 260.48
51.19 123.10 65.12
142.01* 341.49* 180.64*
*
Significant at 5% level.
Y ¼ 80:153 þ 0:995X1 þ 0:523X2 þ 0:360X3 5:594X12 2:540X1 X2 11:154X22 3:725X1 X3 þ 1:125X2 X3 7:339X32
(1)
X1: citric acid (g/kg); X2: glutamate (g/kg); X3: NH4NO3 (g/ kg); Y: PGA (g/kg). The fitted model was checked by the R2, which was evaluated to be 0.9977 (Table 4), it suggested that the model was very adequate in approximating the response surface of the experimental design. The statement was further supported by the test statistics (Table 4), F-value for the overall regression was significant at 5% level and the lack of fit was insignificant. According to the factor static statistical analysis (Table 5), citric acid, glutamate and NH4NO3 had the prominent positive influence on PGA production and their effect decreased in the order of glutamate, NH4NO3 and citric acid. 3D plots of the statistical results are shown in Fig. 6. The maximum point (18.50, 40.14 and 20.04) was resulted in the canonical analysis of response surface, which meant that the optimum composition of the supplementary carbon and nitrogen sources (citric acid: 18.50 g/kg, glutamate: 40.14 g/kg and NH4NO3: 20.04 g/kg) were attained. In the confirmed experiments, the production of PGA reached 83.61 g/kg, which was four times more than the initial output.
The parameters for PGA production in SSF were optimized in this study. The maximum productivity of PGA was 83.61 g/kg where the substrates were composed of soybean cake powder and wheat bran (11:9, w/w), the initial moisture was 65%, the incubation temperature and culture period were 40 8C and 42 h, respectively, the concentration of the supplementary glutamate, citric acid and NH4NO3 were at 40.14 g/kg, 18.50 g/kg and 20.05 g/kg, respectively, in addition to four kinds of mineral salts included MgSO47H2O (0.5 g/kg), CaCl22H2O (0.15 g/kg), FeCl3 6H2O (0.04 g/kg) and MnSO4H2O (0.104 g/kg). SSF process technique might provide a better choice for PGA production than SmF, considering the high productivity and low production cost.
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Optimization of process parameters for poly g-glutamate production under solid state fermentation from Bacillus subtilis CCTCC202048 Xu Jian a,c, Chen Shouwen a,b,c,*, Yu Ziniu a,b,c a National Key Laboratory of Agricultural Microbiology, Wuhan 430070, PR China National Engineering Research Center of Microbial Pesticides, Wuhan 430070, PR China c Department of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, PR China b
Received 20 October 2004; received in revised form 2 February 2005; accepted 14 March 2005
Abstract The solid state fermentation (SSF) was carried out for the production of poly g-glutamate (PGA) from a new strain Bacillus subtilis CCTCC202048. The fermentation factors, which included composition of substrates, initial moisture content, incubation temperature, fermentation period, and additional nutrients such as mineral salts, carbon sources and nitrogen sources, were investigated and optimized. The maximum PGA production (83.61 g/kg of dry substrate) was obtained in the mixed substrates of soybean cake powder and wheat bran (11:9, w/w) supplemented with glutamate (40.14 g/kg), citric acid (18.50 g/kg), NH4NO3 (20.05 g/kg) and mineral salts (MgSO47H2O, CaCl22H2O, FeCl36H2O and MnSO4H2O), with initial moisture content as 65%, at 40 8C incubated for 42 h. This yield was four times more than that before optimization. This is the first report on optimization of PGA production in SSF. # 2005 Elsevier Ltd. All rights reserved. Keywords: Poly g-glutamate (PGA); Solid state fermentation; Bacillus subtilis; Optimization; Response surface experiment; Canonical analysis
1. Introduction Poly g-glutamate (PGA) is an unusual anionic, watersoluble and high viscous polypeptide in which glutamate is polymerized via g-amide linkage. It was first discovered in the capsule of B. anthracis and later found in the cell surrounding of other nonpathogenic Bacillus spp. including B. licheniformis, B. megaterium, Bacillus subtilis, and B. amyloliquefaciens [1]. For its excellent characters such as high efficient water-absorption, completely biodegradable and non-toxic to human, PGA has attracted a variety of interest in the past decade and proved to be a safe and environmental friendly material that has potential applications in a wide range of industry and agriculture such as food, cosmetics, medicine and manure [2,3]. PGA and its derivatives can be used as thickener, humectant, drug carrier, heavy metal absorber, feed additives [4–8], etc. Even though the strategies for the production of PGA in submerged fermentation (SmF) have been researched by several groups * Corresponding author. Tel.: +86 27 87286972; fax: +86 27 87393882. E-mail address: [email protected] (C. Shouwen). 1359-5113/$ – see front matter # 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2005.03.011
[9–11], the fermentation technology for PGA still remains difficult in respect that the significant increase in viscosity of the media results in uncontrollable foaming, limitation of the volumetric oxygen mass transfer which leads to insufficient cell growth and a decrease in PGA yield [12]. Furthermore, a relative high expenditure for SmF media hampers PGA becoming a prevalent commercial valuable product. Solid state fermentation (SSF) is an important mode of fermentation that microorganisms grow on or within the substrates or supports, in the absence or near-absence of free water and excrete aimed products efficiently [13,14]. Considering the lower energy requirement, simplicity of cultivation equipment and high product yield, this traditional method ignites the interest of researchers again recently and has been widely employed in the production of enzymes, fine chemicals, antibiotics [15–17], etc. PGA can be produced by microorganism under SSF since there is a Japanese traditional fermented food Natto made from soybeans by B. subtilis possessing of viscous materials which were mainly composed of PGA. SSF offers an alternative to solve many troubles encountered in SmF for the production of PGA. Firstly, the uncontrollable foaming
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in SmF does not appear in SSF, which will relieve the complexity of controls demanded in SmF to a great extent. Secondly, the expenditure for the media and energy is going to be reduced largely because the cheaper substrates usually composed of agro-industry byproducts or residuals under SSF are used and static cultivation is achieved. Reports on PGA production by SSF are lacking in the literature. In the present study, the aim was to investigate the production of PGA in SSF and achieve a series of optimal process parameters. A newly isolated strain B. subtilis CCTCC202048 that could produce PGA in SmF was employed in this study. The factors considered critical to PGA production in SSF were studied and optimized in this paper.
2. Materials and methods 2.1. Microorganisms and inoculum preparation A new strain of B. subtilis CCTCC202048, isolated from soil samples and developed in this laboratory, was used in the present study. It was maintained on LB agar (tryptone, 10 g/l; yeast extract, 5 g/l; NaCl, 10 g/l; Agar 15 g/l; pH 7.0), stored at 4 8C, and subcultured every 4 weeks. A loopful of the stored strain was transferred to 50 ml LB liquid medium (tryptone, 10 g/l; yeast extract, 5 g/l; NaCl, 10 g/l; pH 7.0), which was incubated in 250 ml flask at 200 rpm at 37 8C. When the turbidity of the culture reached about 4.0 at 600 nm (6 109 cells/ml), cells were harvested by centrifugation at 8000 rpm for 10 min, washed with 0.85% NaCl solution, and then suspended in 50 ml of 0.85% NaCl solution. 2.2. Optimization studies for PGA production in SSF
to the bran was 1:1 (w/w) and the temperature of fermentation was kept at 37 8C. Initial moisture of substrates was adjusted to 50%. The yield of PGA was determined at 36 h. After choosing the best powder substrate, the proportion (8:12, 9:11, 10:10, 11:9, 12:8, 13:7, w/w) between the chosen powder and the wheat bran was optimized too. 2.2.2. Effect of initial moisture content The moisture content of the substrates was adjusted by adding distilled water before autoclaving. The effect of initial moisture content (50%, 55%, 60%, 65%, 70%, w/w) was investigated. The other process parameters were kept identically in this study. 2.2.3. Effect of incubation temperature and period The yields of PGA in six batches fermentation conducted at different temperature (30 8C, 33 8C, 37 8C, 40 8C, 43 8C) were investigated from 6 h to 72 h at an interval of 6 h. The other conditions were kept at their optimized level. 2.2.4. Effect of additional mineral salts Different mineral salts such as MgSO47H2O, CaCl22H2O, FeCl36H2O and MnSO4H2O were dissolved separately in distilled water used to adjust the moisture of the solid substrates. According to previous studies on the effect of salts on PGA production in SmF by B. subtilis [18], the final concentrations for each additional salt in solid media were set to be 0.5 g/kg, 0.15 g/kg, 0.04 g/kg and 0.104 g/kg, respectively, by the order mentioned above. The yield of PGA was determined to study their effect on the final product. 2.2.5. Effect of additional carbon sources Different carbon sources: glucose, lactose, sucrose, starch, citric acid, glycerol and glutamate were added into the solid substrate separately to a final concentration of 20 g/ kg for testing their effect on the yield of PGA.
The static experiments were conducted in 250 ml flasks containing 20 g (dry weight) solid substrates and proper volume of distilled water or nutritious solution, which were sterilized at 121 8C for 30 min prior to fermentation. The initial pH was adjusted to 7.0 with NaOH and inoculum level was 5%. The parameters optimized were as follows: selection of solid substrate and their ratio, initial moisture content, temperature, incubation time, additional carbon sources, additional nitrogen sources and salts additives. The strategy adopted for optimization of the above parameters was consecutive evaluation. Initially one parameter was evaluated and then it was incorporated at its optimized level in the subsequent experiments. The unit ‘g/kg’ quoted in this paper denotes theweight of component per kilogram of dry substrate.
2.2.6. Effect of additional nitrogen sources Different nitrogen sources: NH4Cl, NaNO3, NH4NO3, peptone and yeast extract were added to the solid substrate separately for testing their effect on the yield of PGA. A consistent amount of additive elemental nitrogen (5 g/kg) was needed in this experiment, because the consumption efficient of different type of nitrogen sources would be identified by doing so. The concentration of each additional supplementary is shown in Table 2 according to the content of elemental nitrogen in different nitrogen sources. And when this experiment was conducted, solid substrates were not supplemented with additional carbon sources.
2.2.1. Effect of different solid substrates and their ratio Different effects of four kinds of substrates (the powder of soybean cake, rapeseed cake, cottonseed cake and peanut cake were separately mixed with wheat bran) on PGA production were studied. The proportion of the cake powder
2.2.7. Combination of optimized components Effects of optimized components on PGA production were studied by supplementing combinations of the additional carbon sources components and additional nitrogen sources components chosen in the foregoing two
X. Jian et al. / Process Biochemistry 40 (2005) 3075–3081
experiments. The response surface experiment was conducted for optimizing the concentration of each component using Box-Behnken design in this step. SAS, version 8.01 (Institute Inc., Cary, NC, USA) was used for the experimental design and regression analysis of the experimental data obtained. Canonical analysis was one part of the process RSREG output.
Table 1 Effect of different substrates on PGA production Substrate mixed with wheat brana
PGA production (g/kg)b
Soybean cake powder Rapeseed cake powder Cottonseed cake powder Peanut cake powder
17.89 8.73 7.94 13.45
a b
2.3. PGA extraction
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The ratio of wheat bran and other powder is 1:1. g/kg indicates g PGA/kg dry substrate.
A weighted quantity of fresh fermented substrates were treated in distilled water (1:10, w/v) in flasks and mixed thoroughly on a shaker at 200 rpm for 1 h. The mixtures were filtered through two-layer muslin cloth and the obtained extracts were centrifuged at 12,000 rpm for 15 min. Clarified supernatants were collected for PGA determination. 2.4. PGA determination PGA concentration was detected using the method of Do et al. [19] by HPLC. The quantification system equipped with Waters 515 HPLC pump, Waters 2487 dual l absorbance detector (Milford, MA), and TSK-GEL G6000PWXL column (Tosho, Tokyo, Japan). A mixture of 25 mM Na2SO4 solution-acetronitrile (4:1, v/v) was used as a mobile phase at a flow rate of 0.6 ml/min.
3. Results and discussion 3.1. Optimization of different solid substrates and their ratio The selection of a suitable substrate is a critical factor for a SSF process. There were three points as following needed to be considered: (1) Substrate is not only nutrient material for growth of microorganisms, but also the supporter for microorganisms, giving enough room for oxygen transfer and heat dispersion. (2) In prior research, it was observed that high protein content material was a perfect substrate for B. subtilis CCTCC202048 producing PGA. (3) Wheat bran, one part of carbon sources, can be a satisfying supporter and carrier because of its porosity and cheapness. According to these points above, the initial composition of substrate was designed like this: making the high protein content material powdery, for the sake of increasing substrate consuming rate, and mixing the powder with wheat bran in proper proportion. In the four tested high protein content agro-industrial residues, the powder of soybean cake media proved superior to the other media. The results are shown in Table 1. The ratio between soybean cake powder and wheat bran is an important factor, because overabundance of powder would decrease the porosity of media. To select an optimized ratio, seven kinds of combination were investigated. The highest PGA production was 19.30 g/kg of dry substrate as the results shown (Fig. 1),
Fig. 1. Effect of substrates ratio on PGA production (WP, weight of soybean cake power; WB, weight of wheat bran).
while the ratio between soybean cake powder and wheat bran was 11:9 (w/w). 3.2. Optimization of initial moisture content The initial moisture level is one of the most important factors in SSF media. Five moisture levels ranging from 50% to 70% were established to study their effect on PGA production and the results obtained are shown in Fig. 2. Significantly, the highest production was attained when the initial moisture levels were 60%–65%. Either low or high initial moisture would decrease the final product for the reason that low moisture substrates reduce mass transfer process and high moisture substrates reduce the porosity of the wheat bran [17]. 3.3. Optimization of incubation temperature and period On accounting of mutual interaction affecting the production of PGA between the fermentation temperature and period, the concentrations of PGA in solid media have been determined at an interval of 6 h from 6 h to 72 h at different incubation temperature conditions. Results are shown in Fig. 3. The highest production of PGA was obtained at 40 8C after 42 h. Despite high temperature the moisture content of medium was maintained well during fermentation. It was 54% at 42 h while the initial moisture
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Fig. 2. Effect of initial moisture content on PGA production.
content was 60%. The reason was that enough water was kept by PGA, which had the character of high waterabsorbance [5]. In addition, the end-point of fermentation should be carefully controlled because synthesized PGA would be biodegraded by depolymerase that reported by King et al. [20] at the later stationary phase. At the fermentation temperature of 40 8C, the maximum concentration of PGA in media reached 39.50 g/kg at 42 h and decreased sharply after 48 h. 3.4. Optimization of single additional nutrients The strain produced more PGA caused by each additional salt compared to the production without additional salt as the results shown in Fig. 4. These salts may have the positive effect on enzyme reaction as the composition of co-enzyme in the metabolism of the strain, thus they increased the yield of PGA [2,18,21].
Fig. 3. Effect of incubation temperature and period on PGA production.
Fig. 4. Effect of supplementary mineral salts on PGA production (1, MgSO47H2O; 2, CaCl22H2O; 3, FeCl36H2O; 4, MnSO4H2O; 5, control).
In the seven kinds of carbon sources which were added into solid substrates, glutamate and citric acid enhanced the PGA production by 31.0% and 20.0%, which reached 59.20 g/kg and 54.21 g/kg, respectively. The results are shown in Fig. 5. It was deduced that additional glutamate was material for synthesizing PGA as Ogawa reported in B. subtilis MR-141 [22]. Additional citric acid could be transferred to a-ketoglutaric by the TCA cycle and the aketoglutaric was then converted to glutamate through the glutamate synthetic pathway. This part glutamate was also an important source for consisting PGA unit as described by Goto and Kunioca in B. subtilis IFO3335 [23]. According to those reports and results, it was presumed that the glutamate units of PGA derived from both additional glutamate and the
Fig. 5. Effect of supplementary carbon sources on PGA production (1, glucose; 2, lactose; 3, sucrose; 4, starch; 5, citric acid; 6, glycerol; 7, glutamate; 8, control).
X. Jian et al. / Process Biochemistry 40 (2005) 3075–3081 Table 2 Effect of supplementary nitrogen sources on PGA production
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3.5. Optimization of multiple additional nutrients
Supplementary nitrogen sources
Supplementary concentration (g/kg)a
PGA production (g/kg)
NH4Cl NaNO3 NH4NO3 Peptone Yeast extract Control
19.11 30.36 14.29 41.67 28.57 0
53.56 55.81 59.96 50.23 49.30 44.48
a The Supplementary concentration of elemental nitrogen was unified to 5 g/kg according to corresponding concentration in different nitrogen sources.
glutamate synthesized by glutamate synthetic pathway in this strain. In contrast, glucose, lactose, sucrose and starch were not considered as ideal additional nutrients for that they reduced the PGA production to a great extent. Three kinds of inorganic nitrogen sources (NH4Cl, NaNO3 and NH4NO3) and two kinds of organic nitrogen sources (peptone and yeast extract) were investigated about their effect on PGA production while the additional carbon sources were absent. As the results shown (Table 2), all the nitrogen sources could promote the final product, but the inorganic nitrogen sources were more efficient. The maximum yield of PGA (59.69 g/kg) was obtained by additional NH4NO3 in results. NO3 was observed enhancing the production of PGA more efficiently than NH4+ in this strain in comparing the effect of additional NH4Cl and NaNO3. This character has not been reported in earlier research on PGA production. Kunioka reported that (NH4)2SO4 was assimilated by B. subtilis IFO3335 as a substrate to produce glutamate, which then was converted into PGA [24]. So it was presumed that B. subtilis CCTCC202048 could utilize the additional NO3 and NH4+ as substrates to provide ammonia for synthesizing glutamate and then give a positive effect on PGA production.
Glutamate, citric acid and NH4NO3 were selected as the optimal additional carbon sources and nitrogen sources in the foregoing experiments. Because these kinds of nutrients had shown great efficiency to enhance the PGA production, their additive amounts were worthy to be confirmed. Considering that these three components may have mutual effects on PGA production, they were combined and investigated using Box-Behnken design whose results could be fitted with a second-order polynomial equation by a multiple regression analysis and locate the optimum concentrations in a rapid way. Four kinds of mineral salts were all added to medium, but were not included as factors in this statistical experiment. The reasons were that salts showed insignificant mutual effects with additional glutamate, citric acid and NH4NO3 and their optimum concentration was coincide with the original ones given in the single nutritious experiments (these data are not shown). The experimental design is shown in Table 3, together with the observed data and the predicted values from the model equation. The second-order polynomial equation obtained for PGA production is shown in Eq. (1).
Table 4 ANOVA table of Box-Behnken design experiments Factor
d.f.
SS
R2
F-value
Total model Linear Quadratic Cross product
9 3 3 3
781.08 11.14 683.57 86.37
0.9977 0.0142 0.8731 0.1103
240.74* 10.30* 632.05* 79.86*
Residual Lack of fit Pure error
3 2
1.66 0.14
Total SS
5
1.80
*
7.89
Significant at 5% level.
Table 3 Experimental design and results of the Box-Behnken design together with predicted yield from the model equation Run number
Citric acid (g/kg)
Glutamate (g/kg)
NH4NO3 (g/kg)
Observed PGA yield (g/kg)
Predicted PGA yield (g/kg)
1 2 3 4 5 6 7 8 9 10 11 12 13a 14a 15a
12 12 24 24 12 12 24 24 18 18 18 18 18 18 18
30 50 30 50 40 40 40 40 30 30 50 50 40 40 40
20 20 20 20 10 30 10 30 10 30 10 30 20 20 20
58.82 65.79 66.10 62.91 62.38 70.28 71.61 64.61 62.19 60.93 60.14 63.38 80.42 80.15 79.89
61.89 62.93 63.88 64.92 65.87 66.59 67.86 66.57 60.78 61.50 61.82 62.54 80.15 80.15 80.15
a
Center point.
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Fig. 6. 3D plots of response surface optimization experiment results: (A) the effect of supplementary citric acid concentration, glutamate concentration and their mutual interaction on PGA production. The other factor was held at central point; (B) the effect of supplementary glutamate concentration, NH4NO3 concentration and their mutual interaction on PGA production. The other factor was held at central point.
Table 5 Factor static statistical analysis from the model equation
4. Conclusions
Factor
d.f.
SS
MS
F-value
Citric acid Glutamate NH4NO3
4 4 4
204.77 492.43 260.48
51.19 123.10 65.12
142.01* 341.49* 180.64*
*
Significant at 5% level.
Y ¼ 80:153 þ 0:995X1 þ 0:523X2 þ 0:360X3 5:594X12 2:540X1 X2 11:154X22 3:725X1 X3 þ 1:125X2 X3 7:339X32
(1)
X1: citric acid (g/kg); X2: glutamate (g/kg); X3: NH4NO3 (g/ kg); Y: PGA (g/kg). The fitted model was checked by the R2, which was evaluated to be 0.9977 (Table 4), it suggested that the model was very adequate in approximating the response surface of the experimental design. The statement was further supported by the test statistics (Table 4), F-value for the overall regression was significant at 5% level and the lack of fit was insignificant. According to the factor static statistical analysis (Table 5), citric acid, glutamate and NH4NO3 had the prominent positive influence on PGA production and their effect decreased in the order of glutamate, NH4NO3 and citric acid. 3D plots of the statistical results are shown in Fig. 6. The maximum point (18.50, 40.14 and 20.04) was resulted in the canonical analysis of response surface, which meant that the optimum composition of the supplementary carbon and nitrogen sources (citric acid: 18.50 g/kg, glutamate: 40.14 g/kg and NH4NO3: 20.04 g/kg) were attained. In the confirmed experiments, the production of PGA reached 83.61 g/kg, which was four times more than the initial output.
The parameters for PGA production in SSF were optimized in this study. The maximum productivity of PGA was 83.61 g/kg where the substrates were composed of soybean cake powder and wheat bran (11:9, w/w), the initial moisture was 65%, the incubation temperature and culture period were 40 8C and 42 h, respectively, the concentration of the supplementary glutamate, citric acid and NH4NO3 were at 40.14 g/kg, 18.50 g/kg and 20.05 g/kg, respectively, in addition to four kinds of mineral salts included MgSO47H2O (0.5 g/kg), CaCl22H2O (0.15 g/kg), FeCl3 6H2O (0.04 g/kg) and MnSO4H2O (0.104 g/kg). SSF process technique might provide a better choice for PGA production than SmF, considering the high productivity and low production cost.
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