Rhamsan gum production by Sphingomonas sp. CGMCC 6833 using a two-stage agitation speed control strategy
Xiao Ying Xu1,2,3 Ping Zhu1,2 Sha Li1,2 Xiao Ye Chen1,2 Xing Huan Jiang4 Hong Xu1,2∗
1 State
Key Laboratory of Materials-Oriented Chemical Engineering, Nanjing University of Technology, Nanjing, People’s Republic of China
2 College
of Food Science and Light Industry, Nanjing University of Technology, Nanjing, People’s Republic of China
3 College
of Food Science and Engineering, Nanjing University of Finance and Economics, Nanjing, People’s Republic of China
4 College
of International Education, Nanjing University of Technology, Nanjing, People’s Republic of China
Abstract Varying the agitation speed could greatly affect rhamsan gum production by Sphingomonas sp. CGMCC 6833. Batch fermentations at agitation speeds of 400, 600, 800, and 1,000 rpm were therefore carried out. The time course of specific cell growth rate, specific glucose consumption rate, and specific rhamsan gum formation rate was subsequently determined. Based on the results, a novel two-stage agitation speed control strategy was developed. From 0 to 13 H, the high specific cell growth and glucose consumption rates were achieved by setting the agitation speed of the fermenter at
800 rpm. From 13 H onward to the end of fermentation, the glucose consumption rate and specific cell growth rate were high at the agitation speed of 600 rpm. Using this method, the maximum concentration and productivity of rhamsan gum reached 21.63 ± 1.76 g L−1 and 0.338 ± 0.028 g L−1 H−1 , respectively, which were both higher than the optimum results obtained at constant agitation speeds. C 2013 International Union of Biochemistry and Molecular Biology, Inc. Volume 61, Number 4, Pages 453–458, 2014
Keywords: agitation speed control, batch fermentation, rhamsan gum, Sphingomonas sp. CGMCC 6833, two-stage strategy
1. Introduction The Sphingomonas species produces sphingan, which is composed of water-soluble exopolysaccharides (EPS), such as gellan gum, welan gum, rhamsan gum, S-657, and S-88 [1]. The general backbone structure of EPS is based on a linear
Abbreviations: EPS, exopolysaccharides; DO, dissolved oxygen; μ, specific cell growth rate; qs , specific glucose consumption rate; qp , specific rhamsan gum formation rate; Cx , cell growth; Cs , residual glucose concentration; Cp , rhamsan gum production. ∗ Address for correspondence: Dr. Hong Xu, College of Food Science and Light Industry, Nanjing University of Technology, Nanjing, No. 30 Puzhu South Road, Pukou District, Nanjing, Mail Code 211816, People’s Republic of China. Tel.: +86 025 58139433; Fax: +86 025 58139433; e-mail: [email protected]. Received 26 August 2013; accepted 30 November 2013
DOI: 10.1002/bab.1185 Published online 18 March 2014 in Wiley Online Library (wileyonlinelibrary.com)
tetrasaccharide repeating unit that consists of β-d-glucose, β-d-glucuronic acid, and α-l-rhamnose. Sphingans are widely used as thickening, stabilizing, and suspending agents in pharmaceutical [2], food [3, 4], oil field [5, 6], and concrete [7, 8] industries because these substances are highly viscous and stable. Rhamsan gum has the same backbone structure, and each d-glucosyl residue next to the l-rhamnosyl residue is substituted at O-6 by α-d-glucosyl-(1→6)-β-d-glucosyl disaccharide side chains. The unique structure endows rhamsan gum features favoring industrial applications. Under temperatures higher than 100 ◦ C, rhamsan gum can still maintain its thermostability and high viscosity. Furthermore, rhamsan gum has broader applications in the food industry in comparison to other sphingans because it can tolerate high phosphate and sodium chloride concentrations [9]. The use of rhamsan gum as a food additive was approved by the Japanese Ministry of Health and Welfare in 1996 [10]. As a good anionic water-soluble EPS, rhamsan gum is also safe to use in plastic surgeries [11].
453
Biotechnology and Applied Biochemistry Rhamsan gum is now produced commercially by the CP-Kelco Company. Various types of bioreactors have been used to produce EPS; for instance, a stirred tank is one of the most frequently used bioreactors [12]. Batch fermentation can be significantly influenced by various physical and chemical parameters. Among them, agitation speed is an important parameter, especially in stirred tanks because speed can affect the rate of oxygen mass transfer, thereby influencing the fermentation rate and fermented metabolite production. An instance of this is that limited oxygen at low agitation speeds resulted in lower specific xanthan production rates. Higher rates were obtained at 1,000 rpm at 50 H of fermentation [13, 14]. Studies have also shown that a higher agitation speed could yield higher biomass but not higher EPS production rate during sphingan fermentation. An example would be welan gum production [15]. It would be interesting to see which is affected by agitation speed during rhamsan gum production. In this study, rhamsan gum production was enhanced by using the method of agitation speed control. Rhamsan gum fermentation by Sphingomonas sp. CGMCC 6833 was investigated under different agitation speeds in a 7.5 L fermenter. Based on the batch fermentation kinetic analysis, a two-stage agitation speed control strategy was proposed and experimentally confirmed to achieve both a high yield and high production rate of rhamsan gum.
2. Materials and Methods 2.1. Microorganism and media Sphingomonas sp. RH-1 (CGMCC 6833), a rhamsan gum– producing strain, was originally isolated from a soil sample collected from Laoshan National Forest Park of Nanjing (Nanjing, People’s Republic of China) [16]. The seed medium was composed of glucose 20 g L−1 , yeast extract 1 g L−1 , peptone 3 g L−1 , K2 HPO4 2 g L−1 , MgSO4 0.1 g L−1 and pH 7.5. The batch fermentation medium was composed of glucose 40 g L−1 , yeast extract 5.4 g L−1 , K2 HPO4 5.7 g L−1 , MnSO4 0.3 g L−1 , and pH 7.5.
2.2. Culture methods Sphingomonas sp. CGMCC 6833 was inoculated in 100 mL of fresh seed medium in 500 mL flasks and cultivated on a shaker at 200 rpm and 30 ◦ C for 16 H. The seed culture (5%, v/v) was then inoculated into the fermentation medium. Batch fermentation was carried out in a 7.5 L stirred fermenter (Rushton impeller; BioFlo110; New Brunswick Scientific, Edison, NJ, USA) with a working volume of 4.5 L. Sphingomonas sp. CGMCC 6833 was cultivated at 30 ◦ C. The aeration rate was controlled at 1.0 volume of liquid per Min, and pH was maintained at 7.5 automatically by adding 6 mol L−1 of NaOH or 6 mol L−1 of HCl. The agitation speed was controlled at 400, 600, 800, and 1,000 rpm in fermentation, respectively. At the fermentation temperature
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of 30 ◦ C, 100% dissolved oxygen (DO) saturation corresponded to an actual DO tension of approximately 7.54 mg L−1 at 1.0 atm. Under the fermentation condition, the oxygen transfer coefficient kL a under each agitation speed was measured by the sulfite oxidation method of using a Na2 SO3 solution. The oxygen uptake (OUR) was determined using the dynamic method [17]. The effects of different agitation speeds on biomass, rhamsan gum production, glucose consumption, and viscosity of fermentation broth were studied at the different fermentation conditions.
2.3. Analytical methods Ten milliliters of cell suspensions was harvested by centrifugation at 12,000g for 30 Min, washed with distilled water, then dried in an oven at 60 ◦ C for 24 H to obtain a constant weight to determine the biomass. The concentration of glucose was measured by using a biosensor (SBA-40C; Shandong Academy of Sciences, Jinan, Shandong, People’s Republic of China) [18]. The viscosity of the fermentation broth was measured by using a rotational viscometer (NDJ-1; Shanghai Hengping Scientific Instrument Company, Shanghai, People’s Republic of China) with rotor No. 3 at 30 rpm. pH was measured using a precise pH meter (Shanghai Leici Instrument Co., Ltd., Shanghai, People’s Republic of China). To measure the concentration of rhamsan gum, the following procedures were performed: A certain volume of fermentation broth was heated in a water bath at 80 ◦ C for 15 Min. After cooling, the cells were removed by centrifugation at 12,000g for 30 Min. Double volumes of anhydrous ethanol were added with stirring until a flocculent precipitate appeared. The solution was put in a refrigerator at 4 ◦ C for 12 H. After centrifugation, the supernatant was removed. The process was then repeated using anhydrous ethanol. The precipitate was dried in an oven at 60 ◦ C to obtain a constant weight. The molecular weight of rhamsan gum at different agitation speeds was measured using the Laurent method. The Mark–Houwink constants were k = 3.6 × 10−4 and a = 0.78.
2.4. Calculation of kinetic parameters Based on the experimental or fitted data of cell growth (Cx , g L−1 ), residual glucose concentration (Cs , g L−1 ), and rhamsan gum production (Cp , g L−1 ), the parameters of specific cell growth rate (µ, H−1 ), specific glucose consumption rate (qs , H−1 ), and specific rhamsan gum formation rate (qp , H−1 ) were calculated by using Eqs. 1 to 3, respectively (version 4; Golden Software, Golden, CO, USA). μ=
qs = −
qp =
1 C x 1 dC x = lim C x dt C x t→0 t
(1)
C s 1 dC s 1 lim =− C x dt C x t→0 t
(2)
C p 1 dC p 1 lim = C x dt C x t→0 t
(3)
Rhamsan Gum Production by Sphingomonas sp. CGMCC 6833
FIG. 1
Time course of rhamsan gum production at different agitation speeds: 400 rpm (triangles), 600 rpm (filled circles), 800 rpm (squares), and 1,000 rpm (filled triangles). (A) Biomass, (B) glucose, (C) rhamsan gum, and (D) viscosity.
3. Results and Discussion 3.1. Time course of rhamsan gum fermentation at different agitation speeds The effects of agitation speed on the fermentation of rhamsan gum using Sphingomonas sp. CGMCC 6833 are shown in Fig. 1. During cultivation, agitation speed for the separate batch was maintained at 400, 600, 800, and 1,000 rpm. The results indicated that agitation speed is important in rhamsan gum production. The two highest rhamsan gum concentrations, 18.56 ± 1.78 and 19.82 ± 1.62 g L−1 , were achieved at 600 and 800 rpm, respectively (Figs. 1A–1D), whereas the rhamsan gum productivity at 800 rpm (0.310 ± 0.025 g L−1 H−1 ) was higher than that at 600 rpm (0.290 ± 0.028 g L−1 H−1 ). Either higher or lower agitation speed resulted in decreased maximum rhamsan gum concentrations (14.66 ± 1.29 g L−1 at 400 rpm, 17.59 ± 1.45 g L−1 at 1,000 rpm), suggesting that either low (400 rpm) or high (1,000 rpm) agitation speed could not improve rhamsan gum fermentation. The biomass was positively correlated with agitation speed. It had values of 4.83 ± 0.42, 7.23 ± 0.65, 7.36 ± 0.54, and 7.69 ± 0.68 g L−1 at
Biotechnology and Applied Biochemistry
400, 600, 800, and 1,000 rpm, respectively (Fig. 2A). However, glucose consumption declined as the agitation slowed down, reflecting a decreased cell growth. These observations indicated that the agitation speed optimum for biomass accumulation was not suitable to ensure high rhamsan gum concentration and productivity. When the agitation speed was either too low or too high, it was hard to obtain highly viscous rhamsan gum (Fig. 2D). At the end of fermentation, although the rhamsan gum concentration at 800 rpm was higher than that at 600 rpm, the rhamsan gum viscosity at 800 rpm (3.12 ± 0.31 Pa·Sec) was lower than that at 600 rpm (3.42 ± 0.30 Pa·Sec). This observation may indicate that very high agitation speeds caused mechanical damage to rhamsan gum. Therefore, if the agitation speed is constant, the productivity and viscosity of rhamsan gum could not be simultaneously high during the whole fermentation process. The molecular weight at different agitation speeds is presented in Table 1. The maximum was reached at a speed of 600 rpm. However, higher speed was not beneficial to the molecular weight. When the agitation speed increased to 800 or 1,000 rpm, the molecular weight decreased. Agitation and aeration are related to DO tension and are considered important in sphingan fermentation [19]. DO tension can be controlled more easily when the agitation speed can be altered. The changing patterns of DO tension at different agitation speeds are shown in Fig. 2. DO tension
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Biotechnology and Applied Biochemistry resulted in slow cell growth after 13 H. The DO tension in the stationary phase was maintained at a constant low level. The kL a increased along with the agitation speed (Table 1). The OUR values in the stationary phase were consistent. The increase in OUR values was obtained with the increase in agitation speed. In the process of a oxygen-limited system, DO tension plays an important role [20, 21]. The influence of agitation speed on batch fermentation should be further studied.
3.2. Kinetic analysis of rhamsan gum fermentation at different agitation speeds
Profiles of dissolved oxygen tension in batch production of rhamsan gum using different agitation speed control strategies: 400 rpm (curve 1), 600 rpm (curve 2), 800 rpm (curve 3), 1,000 rpm (curve 4), and two-stage agitation speed control strategy (curve 5).
FIG. 2
achieved the lowest values of 9.3%, 18.2%, 24.1%, and 28.9% of air saturation at 400, 600, 800, and 1,000 rpm, respectively. Generally, a rapid decrease in DO was observed in the first 13 H, after which the DO value was almost constant. During the first 13 H, the DO tension was higher under intensive agitation, which would induce an increase in cell population and a decrease in glucose concentration. As the agitation speed was set to a lower value, the decreasing DO tension
The kinetic characteristics of µ, qs , and qp were calculated by the data obtained at 600 and 800 rpm (Fig. 3). During batch fermentation, µ, qs , and qp exhibited similar trends; the maximum values were observed at approximately 5 H. At the beginning of rhamsan gum fermentation, µ and qs were higher at 800 than at 600 rpm. Thus, 800 rpm was concluded to be more beneficial for cell growth and glucose consumption. After 13 H, however, 600 rpm appeared to be more helpful for rhamsan gum formation, showing that lower agitation speed could result in more rhamsan gum accumulated at later stages of cultivation. Therefore, by analyzing the three key parameters, µ, qs , and qp , a two-stage agitation speed control strategy was formulated. According to this strategy, in the first 13 H, an agitation speed of 800 rpm is helpful to maintain a high value for both µ and qs ; after 13 H, the agitation speed can be reduced to 600 rpm to maintain a high qp .
3.3. Rhamsan gum production with two-stage agitation speed strategy The time course of the proposed two-stage agitation speed strategy and the changing patterns of DO tension for rhamsan
Comparison of parameters in batch production of rhamsan gum by Sphingomonas sp. CGMCC 6833 using different agitation speed control strategiesa
TABLE 1
400 rpm
600 rpm
800 rpm
1,000 rpm
800 rpm (0–13 H), 600 rpm (after 13 H)
4.83 ± 0.42
7.23 ± 0.65
7.36 ± 0.54
7.69 ± 0.68
7.26 ± 0.63
Rhamsan gum (g L )
14.66 ± 1.29
18.56 ± 1.78
19.82 ± 1.62
17.59 ± 1.45
21.63 ± 1.76
Rhamsan gum productivity (g L−1 H−1 )
0.229 ± 0.020
0.290 ± 0.028
0.310 ± 0.025
0.275 ± 0.023
0.338 ± 0.028
Residual glucose (g L−1 )
23.75 ± 1.50
15.25 ± 1.25
14.25 ± 1.25
12.25 ± 1.25
14.75 ± 1.25
Fermentation broth viscosity (Pa·Sec)
2.19 ± 0.20
3.42 ± 0.30
3.12 ± 0.31
2.82 ± 0.28
3.55 ± 0.29
Rhamsan gum molecular weight (×10−5 Da)
8.44 ± 0.09
8.83 ± 0.08
8.69 ± 0.09
8.53 ± 0.11
9.05 ± 0.09
kL a (H−1 )
43.7 ± 2.9
62.3 ± 3.4
75.7 ± 2.7
86.9 ± 3.8
–
7.87 ± 0.52
10.17 ± 0.55
11.37 ± 0.41
12.15 ± 0.53
–
Parameter Biomass (g L−1 ) −1
−1
OUR (mol L a Values
456
−1
H )
are mean ± standard deviation (N = 3).
Rhamsan Gum Production by Sphingomonas sp. CGMCC 6833
FIG. 4
Rhamsan gum fermentation with two-stage agitation speed control strategy. Glucose (filled squares), rhamsan gum (filled circles), biomass (circles), and viscosity (triangles).
improved rhamsan gum production and increased rhamsan gum productivity (Table 1). The maximum concentration and productivity of rhamsan gum reached 21.63 ± 1.76 g L−1 and 0.338 ± 0.028 g L−1 H−1 , respectively, which were higher than the optimum results obtained at constant agitation speed (19.82 ± 1.62 g L−1 of the rhamsan gum concentration and 0.310 ± 0.025 g L−1 H−1 of rhamsan gum productivity at 800 rpm). Using the two-stage agitation speed control strategy, the rhamsan gum fermentation broth viscosity reached 3.55 ± 0.29 Pa·Sec at the end of fermentation, which was higher than the optimum result obtained at 600 rpm. Therefore, this method could remarkably enhance rhamsan gum production.
4. Conclusions
FIG. 3
Comparison of (A) specific growth rate (μ), (B) specific substrate consumption rate (qs), and (C) specific rhamsan gum formation rate (qp ) between 600 rpm (dashed curve) and 800 rpm (solid curve).
gum fermentation are shown in Figs. 4 and 2. A quick reduction in DO tension at 800 rpm from 0 to 13 H was observed. As agitation speed decreased to 600 rpm, DO tension was higher than that at a constant agitation speed of 600 rpm. The relatively high DO tension was favorable for cell growth. The two-stage agitation speed control strategy considerably
Biotechnology and Applied Biochemistry
In this study, a novel two-stage agitation speed control strategy was developed based on the kinetic analysis of rhamsan gum fermentation with Sphingomonas sp. CGMCC 6833. It effectively enhanced rhamsan gum concentration and productivity, as well as fermentation broth viscosity. Compared with the optimum results at the constant agitation speed strategy (800 rpm), rhamsan gum concentration and productivity were enhanced 9.13% and 9.03%, respectively, by applying this method.
5. Acknowledgements This study was financially supported by the National Natural Science Foundation of China (grant nos. 21006050 and 21106062) and the National Key Technology R&D Program (grant no. 2011BAD23B04).
6. References [1] Banik, R. M., Kanari, B., and Upadhyay, S. N. (2000) World J. Microb. Biotechnol. 16, 407–414.
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Biotechnology and Applied Biochemistry [2] Coviello, T., Dentini, M., Rambone, G., Desideri, P., Carafa, M., Murtas, E., Riccieria, F. M., and Alhaiquea, F. (1998) J. Control Release 55, 57–66. [3] Morrison, N. A., Clark, R. C., Chen, Y. L., Talashek, T., and Sworn, G. (1999) Prog. Colloid Polym. Sci. 114, 127–131. [4] Bajaj, I., and Singhal, R. (2007) Food Chem. 104, 1472–1477. [5] Dennis, H. H., Thomas, O. M., and Paul, S. (1991) US Patent 4981520. [6] Vercaemer, C. J., Davies, S. N., Pafitis, D.G., Maintland, G. C., and Poyet, J. P. (1997) US Patent 5697441. [7] Plank, J. (2004) Appl. Microbiol. Biotechnol. 66, 1–9. [8] Allen, F. L., Best, G. H., and Linfroth, T. A. (1991) US Patent 5004506. [9] Kang, K. S., and Pettitt, D. J. (1993) In Industrial Gums: Polysaccharides and Their Derivatives (BeMiller, J. N., and Whistler, R. L., eds.). pp. 341–397, Academic Press, San Diego, CA. [10] Hagiwara, A., Imai, N., Doi, Y., Sano, M., Tamano, S., Omoto, T., Asai, I., Yasuhara, K., and Hayashi, S. (2010) J. Toxicol. Sci. 35, 493–501. [11] Paula, M. D., Goissis, G., and Martins, V. C. A. (2007) J. Mater. Sci. 18, 1683–1690.
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[12] Arockiasamy, S., and Banik, R. M. (2008) J. Biosci. Bioeng. 105, 204–210. [13] Suh, I. S., Herbst, H., Schumpe, A., and Deckwer, W. D. (1990) Biotechnol. Lett. 12, 201–206. [14] Amanullah, A., Satti, S., and Nienow, A. W. (1998) Biotechnol. Prog. 14, 265–269. [15] Li, H., Xu, H., Li, S., Feng, X. H., Xu, H., and Ouyang, P. K. (2011) Process Biochem. 46, 1172–1178. [16] Xu, H., Xu, X. Y., Dong, S. H., Li, S., and Liang, J. F. (2012) China Patent Application 201210504461.3. [17] Shang, F., Wen, S. H., Wang, X., and Tan, T. W. (2006) J. Biosci. Bioeng. 101, 38–41. [18] Xu, H., Jiang, M., Li, H., Lu, D. Q., and Ouyang, P. K. (2005) Process Biochem. 40, 519–523. [19] Banik, R. M., and Santhiagu, A. (2006) Biotechnol. Lett. 28, 1347–1350. [20] Wang, Z. J., Wang, H. Y., Li, Y. L., Chu, J., Huang, M. Z., Zhuang, Y. P., and Zhang, S. L. (2010) Bioresour. Technol. 101, 2845–2852. [21] Xiao, J., Shi, Z. P., Gao, P., Feng, H. J., Duan, Z. Y., and Mao, Z. G. (2006) Bioproc. Biosyst. Eng. 29, 109–117.
Rhamsan Gum Production by Sphingomonas sp. CGMCC 6833
Xiao Ying Xu1,2,3 Ping Zhu1,2 Sha Li1,2 Xiao Ye Chen1,2 Xing Huan Jiang4 Hong Xu1,2∗
1 State
Key Laboratory of Materials-Oriented Chemical Engineering, Nanjing University of Technology, Nanjing, People’s Republic of China
2 College
of Food Science and Light Industry, Nanjing University of Technology, Nanjing, People’s Republic of China
3 College
of Food Science and Engineering, Nanjing University of Finance and Economics, Nanjing, People’s Republic of China
4 College
of International Education, Nanjing University of Technology, Nanjing, People’s Republic of China
Abstract Varying the agitation speed could greatly affect rhamsan gum production by Sphingomonas sp. CGMCC 6833. Batch fermentations at agitation speeds of 400, 600, 800, and 1,000 rpm were therefore carried out. The time course of specific cell growth rate, specific glucose consumption rate, and specific rhamsan gum formation rate was subsequently determined. Based on the results, a novel two-stage agitation speed control strategy was developed. From 0 to 13 H, the high specific cell growth and glucose consumption rates were achieved by setting the agitation speed of the fermenter at
800 rpm. From 13 H onward to the end of fermentation, the glucose consumption rate and specific cell growth rate were high at the agitation speed of 600 rpm. Using this method, the maximum concentration and productivity of rhamsan gum reached 21.63 ± 1.76 g L−1 and 0.338 ± 0.028 g L−1 H−1 , respectively, which were both higher than the optimum results obtained at constant agitation speeds. C 2013 International Union of Biochemistry and Molecular Biology, Inc. Volume 61, Number 4, Pages 453–458, 2014
Keywords: agitation speed control, batch fermentation, rhamsan gum, Sphingomonas sp. CGMCC 6833, two-stage strategy
1. Introduction The Sphingomonas species produces sphingan, which is composed of water-soluble exopolysaccharides (EPS), such as gellan gum, welan gum, rhamsan gum, S-657, and S-88 [1]. The general backbone structure of EPS is based on a linear
Abbreviations: EPS, exopolysaccharides; DO, dissolved oxygen; μ, specific cell growth rate; qs , specific glucose consumption rate; qp , specific rhamsan gum formation rate; Cx , cell growth; Cs , residual glucose concentration; Cp , rhamsan gum production. ∗ Address for correspondence: Dr. Hong Xu, College of Food Science and Light Industry, Nanjing University of Technology, Nanjing, No. 30 Puzhu South Road, Pukou District, Nanjing, Mail Code 211816, People’s Republic of China. Tel.: +86 025 58139433; Fax: +86 025 58139433; e-mail: [email protected]. Received 26 August 2013; accepted 30 November 2013
DOI: 10.1002/bab.1185 Published online 18 March 2014 in Wiley Online Library (wileyonlinelibrary.com)
tetrasaccharide repeating unit that consists of β-d-glucose, β-d-glucuronic acid, and α-l-rhamnose. Sphingans are widely used as thickening, stabilizing, and suspending agents in pharmaceutical [2], food [3, 4], oil field [5, 6], and concrete [7, 8] industries because these substances are highly viscous and stable. Rhamsan gum has the same backbone structure, and each d-glucosyl residue next to the l-rhamnosyl residue is substituted at O-6 by α-d-glucosyl-(1→6)-β-d-glucosyl disaccharide side chains. The unique structure endows rhamsan gum features favoring industrial applications. Under temperatures higher than 100 ◦ C, rhamsan gum can still maintain its thermostability and high viscosity. Furthermore, rhamsan gum has broader applications in the food industry in comparison to other sphingans because it can tolerate high phosphate and sodium chloride concentrations [9]. The use of rhamsan gum as a food additive was approved by the Japanese Ministry of Health and Welfare in 1996 [10]. As a good anionic water-soluble EPS, rhamsan gum is also safe to use in plastic surgeries [11].
453
Biotechnology and Applied Biochemistry Rhamsan gum is now produced commercially by the CP-Kelco Company. Various types of bioreactors have been used to produce EPS; for instance, a stirred tank is one of the most frequently used bioreactors [12]. Batch fermentation can be significantly influenced by various physical and chemical parameters. Among them, agitation speed is an important parameter, especially in stirred tanks because speed can affect the rate of oxygen mass transfer, thereby influencing the fermentation rate and fermented metabolite production. An instance of this is that limited oxygen at low agitation speeds resulted in lower specific xanthan production rates. Higher rates were obtained at 1,000 rpm at 50 H of fermentation [13, 14]. Studies have also shown that a higher agitation speed could yield higher biomass but not higher EPS production rate during sphingan fermentation. An example would be welan gum production [15]. It would be interesting to see which is affected by agitation speed during rhamsan gum production. In this study, rhamsan gum production was enhanced by using the method of agitation speed control. Rhamsan gum fermentation by Sphingomonas sp. CGMCC 6833 was investigated under different agitation speeds in a 7.5 L fermenter. Based on the batch fermentation kinetic analysis, a two-stage agitation speed control strategy was proposed and experimentally confirmed to achieve both a high yield and high production rate of rhamsan gum.
2. Materials and Methods 2.1. Microorganism and media Sphingomonas sp. RH-1 (CGMCC 6833), a rhamsan gum– producing strain, was originally isolated from a soil sample collected from Laoshan National Forest Park of Nanjing (Nanjing, People’s Republic of China) [16]. The seed medium was composed of glucose 20 g L−1 , yeast extract 1 g L−1 , peptone 3 g L−1 , K2 HPO4 2 g L−1 , MgSO4 0.1 g L−1 and pH 7.5. The batch fermentation medium was composed of glucose 40 g L−1 , yeast extract 5.4 g L−1 , K2 HPO4 5.7 g L−1 , MnSO4 0.3 g L−1 , and pH 7.5.
2.2. Culture methods Sphingomonas sp. CGMCC 6833 was inoculated in 100 mL of fresh seed medium in 500 mL flasks and cultivated on a shaker at 200 rpm and 30 ◦ C for 16 H. The seed culture (5%, v/v) was then inoculated into the fermentation medium. Batch fermentation was carried out in a 7.5 L stirred fermenter (Rushton impeller; BioFlo110; New Brunswick Scientific, Edison, NJ, USA) with a working volume of 4.5 L. Sphingomonas sp. CGMCC 6833 was cultivated at 30 ◦ C. The aeration rate was controlled at 1.0 volume of liquid per Min, and pH was maintained at 7.5 automatically by adding 6 mol L−1 of NaOH or 6 mol L−1 of HCl. The agitation speed was controlled at 400, 600, 800, and 1,000 rpm in fermentation, respectively. At the fermentation temperature
454
of 30 ◦ C, 100% dissolved oxygen (DO) saturation corresponded to an actual DO tension of approximately 7.54 mg L−1 at 1.0 atm. Under the fermentation condition, the oxygen transfer coefficient kL a under each agitation speed was measured by the sulfite oxidation method of using a Na2 SO3 solution. The oxygen uptake (OUR) was determined using the dynamic method [17]. The effects of different agitation speeds on biomass, rhamsan gum production, glucose consumption, and viscosity of fermentation broth were studied at the different fermentation conditions.
2.3. Analytical methods Ten milliliters of cell suspensions was harvested by centrifugation at 12,000g for 30 Min, washed with distilled water, then dried in an oven at 60 ◦ C for 24 H to obtain a constant weight to determine the biomass. The concentration of glucose was measured by using a biosensor (SBA-40C; Shandong Academy of Sciences, Jinan, Shandong, People’s Republic of China) [18]. The viscosity of the fermentation broth was measured by using a rotational viscometer (NDJ-1; Shanghai Hengping Scientific Instrument Company, Shanghai, People’s Republic of China) with rotor No. 3 at 30 rpm. pH was measured using a precise pH meter (Shanghai Leici Instrument Co., Ltd., Shanghai, People’s Republic of China). To measure the concentration of rhamsan gum, the following procedures were performed: A certain volume of fermentation broth was heated in a water bath at 80 ◦ C for 15 Min. After cooling, the cells were removed by centrifugation at 12,000g for 30 Min. Double volumes of anhydrous ethanol were added with stirring until a flocculent precipitate appeared. The solution was put in a refrigerator at 4 ◦ C for 12 H. After centrifugation, the supernatant was removed. The process was then repeated using anhydrous ethanol. The precipitate was dried in an oven at 60 ◦ C to obtain a constant weight. The molecular weight of rhamsan gum at different agitation speeds was measured using the Laurent method. The Mark–Houwink constants were k = 3.6 × 10−4 and a = 0.78.
2.4. Calculation of kinetic parameters Based on the experimental or fitted data of cell growth (Cx , g L−1 ), residual glucose concentration (Cs , g L−1 ), and rhamsan gum production (Cp , g L−1 ), the parameters of specific cell growth rate (µ, H−1 ), specific glucose consumption rate (qs , H−1 ), and specific rhamsan gum formation rate (qp , H−1 ) were calculated by using Eqs. 1 to 3, respectively (version 4; Golden Software, Golden, CO, USA). μ=
qs = −
qp =
1 C x 1 dC x = lim C x dt C x t→0 t
(1)
C s 1 dC s 1 lim =− C x dt C x t→0 t
(2)
C p 1 dC p 1 lim = C x dt C x t→0 t
(3)
Rhamsan Gum Production by Sphingomonas sp. CGMCC 6833
FIG. 1
Time course of rhamsan gum production at different agitation speeds: 400 rpm (triangles), 600 rpm (filled circles), 800 rpm (squares), and 1,000 rpm (filled triangles). (A) Biomass, (B) glucose, (C) rhamsan gum, and (D) viscosity.
3. Results and Discussion 3.1. Time course of rhamsan gum fermentation at different agitation speeds The effects of agitation speed on the fermentation of rhamsan gum using Sphingomonas sp. CGMCC 6833 are shown in Fig. 1. During cultivation, agitation speed for the separate batch was maintained at 400, 600, 800, and 1,000 rpm. The results indicated that agitation speed is important in rhamsan gum production. The two highest rhamsan gum concentrations, 18.56 ± 1.78 and 19.82 ± 1.62 g L−1 , were achieved at 600 and 800 rpm, respectively (Figs. 1A–1D), whereas the rhamsan gum productivity at 800 rpm (0.310 ± 0.025 g L−1 H−1 ) was higher than that at 600 rpm (0.290 ± 0.028 g L−1 H−1 ). Either higher or lower agitation speed resulted in decreased maximum rhamsan gum concentrations (14.66 ± 1.29 g L−1 at 400 rpm, 17.59 ± 1.45 g L−1 at 1,000 rpm), suggesting that either low (400 rpm) or high (1,000 rpm) agitation speed could not improve rhamsan gum fermentation. The biomass was positively correlated with agitation speed. It had values of 4.83 ± 0.42, 7.23 ± 0.65, 7.36 ± 0.54, and 7.69 ± 0.68 g L−1 at
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400, 600, 800, and 1,000 rpm, respectively (Fig. 2A). However, glucose consumption declined as the agitation slowed down, reflecting a decreased cell growth. These observations indicated that the agitation speed optimum for biomass accumulation was not suitable to ensure high rhamsan gum concentration and productivity. When the agitation speed was either too low or too high, it was hard to obtain highly viscous rhamsan gum (Fig. 2D). At the end of fermentation, although the rhamsan gum concentration at 800 rpm was higher than that at 600 rpm, the rhamsan gum viscosity at 800 rpm (3.12 ± 0.31 Pa·Sec) was lower than that at 600 rpm (3.42 ± 0.30 Pa·Sec). This observation may indicate that very high agitation speeds caused mechanical damage to rhamsan gum. Therefore, if the agitation speed is constant, the productivity and viscosity of rhamsan gum could not be simultaneously high during the whole fermentation process. The molecular weight at different agitation speeds is presented in Table 1. The maximum was reached at a speed of 600 rpm. However, higher speed was not beneficial to the molecular weight. When the agitation speed increased to 800 or 1,000 rpm, the molecular weight decreased. Agitation and aeration are related to DO tension and are considered important in sphingan fermentation [19]. DO tension can be controlled more easily when the agitation speed can be altered. The changing patterns of DO tension at different agitation speeds are shown in Fig. 2. DO tension
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3.2. Kinetic analysis of rhamsan gum fermentation at different agitation speeds
Profiles of dissolved oxygen tension in batch production of rhamsan gum using different agitation speed control strategies: 400 rpm (curve 1), 600 rpm (curve 2), 800 rpm (curve 3), 1,000 rpm (curve 4), and two-stage agitation speed control strategy (curve 5).
FIG. 2
achieved the lowest values of 9.3%, 18.2%, 24.1%, and 28.9% of air saturation at 400, 600, 800, and 1,000 rpm, respectively. Generally, a rapid decrease in DO was observed in the first 13 H, after which the DO value was almost constant. During the first 13 H, the DO tension was higher under intensive agitation, which would induce an increase in cell population and a decrease in glucose concentration. As the agitation speed was set to a lower value, the decreasing DO tension
The kinetic characteristics of µ, qs , and qp were calculated by the data obtained at 600 and 800 rpm (Fig. 3). During batch fermentation, µ, qs , and qp exhibited similar trends; the maximum values were observed at approximately 5 H. At the beginning of rhamsan gum fermentation, µ and qs were higher at 800 than at 600 rpm. Thus, 800 rpm was concluded to be more beneficial for cell growth and glucose consumption. After 13 H, however, 600 rpm appeared to be more helpful for rhamsan gum formation, showing that lower agitation speed could result in more rhamsan gum accumulated at later stages of cultivation. Therefore, by analyzing the three key parameters, µ, qs , and qp , a two-stage agitation speed control strategy was formulated. According to this strategy, in the first 13 H, an agitation speed of 800 rpm is helpful to maintain a high value for both µ and qs ; after 13 H, the agitation speed can be reduced to 600 rpm to maintain a high qp .
3.3. Rhamsan gum production with two-stage agitation speed strategy The time course of the proposed two-stage agitation speed strategy and the changing patterns of DO tension for rhamsan
Comparison of parameters in batch production of rhamsan gum by Sphingomonas sp. CGMCC 6833 using different agitation speed control strategiesa
TABLE 1
400 rpm
600 rpm
800 rpm
1,000 rpm
800 rpm (0–13 H), 600 rpm (after 13 H)
4.83 ± 0.42
7.23 ± 0.65
7.36 ± 0.54
7.69 ± 0.68
7.26 ± 0.63
Rhamsan gum (g L )
14.66 ± 1.29
18.56 ± 1.78
19.82 ± 1.62
17.59 ± 1.45
21.63 ± 1.76
Rhamsan gum productivity (g L−1 H−1 )
0.229 ± 0.020
0.290 ± 0.028
0.310 ± 0.025
0.275 ± 0.023
0.338 ± 0.028
Residual glucose (g L−1 )
23.75 ± 1.50
15.25 ± 1.25
14.25 ± 1.25
12.25 ± 1.25
14.75 ± 1.25
Fermentation broth viscosity (Pa·Sec)
2.19 ± 0.20
3.42 ± 0.30
3.12 ± 0.31
2.82 ± 0.28
3.55 ± 0.29
Rhamsan gum molecular weight (×10−5 Da)
8.44 ± 0.09
8.83 ± 0.08
8.69 ± 0.09
8.53 ± 0.11
9.05 ± 0.09
kL a (H−1 )
43.7 ± 2.9
62.3 ± 3.4
75.7 ± 2.7
86.9 ± 3.8
–
7.87 ± 0.52
10.17 ± 0.55
11.37 ± 0.41
12.15 ± 0.53
–
Parameter Biomass (g L−1 ) −1
−1
OUR (mol L a Values
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−1
H )
are mean ± standard deviation (N = 3).
Rhamsan Gum Production by Sphingomonas sp. CGMCC 6833
FIG. 4
Rhamsan gum fermentation with two-stage agitation speed control strategy. Glucose (filled squares), rhamsan gum (filled circles), biomass (circles), and viscosity (triangles).
improved rhamsan gum production and increased rhamsan gum productivity (Table 1). The maximum concentration and productivity of rhamsan gum reached 21.63 ± 1.76 g L−1 and 0.338 ± 0.028 g L−1 H−1 , respectively, which were higher than the optimum results obtained at constant agitation speed (19.82 ± 1.62 g L−1 of the rhamsan gum concentration and 0.310 ± 0.025 g L−1 H−1 of rhamsan gum productivity at 800 rpm). Using the two-stage agitation speed control strategy, the rhamsan gum fermentation broth viscosity reached 3.55 ± 0.29 Pa·Sec at the end of fermentation, which was higher than the optimum result obtained at 600 rpm. Therefore, this method could remarkably enhance rhamsan gum production.
4. Conclusions
FIG. 3
Comparison of (A) specific growth rate (μ), (B) specific substrate consumption rate (qs), and (C) specific rhamsan gum formation rate (qp ) between 600 rpm (dashed curve) and 800 rpm (solid curve).
gum fermentation are shown in Figs. 4 and 2. A quick reduction in DO tension at 800 rpm from 0 to 13 H was observed. As agitation speed decreased to 600 rpm, DO tension was higher than that at a constant agitation speed of 600 rpm. The relatively high DO tension was favorable for cell growth. The two-stage agitation speed control strategy considerably
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In this study, a novel two-stage agitation speed control strategy was developed based on the kinetic analysis of rhamsan gum fermentation with Sphingomonas sp. CGMCC 6833. It effectively enhanced rhamsan gum concentration and productivity, as well as fermentation broth viscosity. Compared with the optimum results at the constant agitation speed strategy (800 rpm), rhamsan gum concentration and productivity were enhanced 9.13% and 9.03%, respectively, by applying this method.
5. Acknowledgements This study was financially supported by the National Natural Science Foundation of China (grant nos. 21006050 and 21106062) and the National Key Technology R&D Program (grant no. 2011BAD23B04).
6. References [1] Banik, R. M., Kanari, B., and Upadhyay, S. N. (2000) World J. Microb. Biotechnol. 16, 407–414.
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Biotechnology and Applied Biochemistry [2] Coviello, T., Dentini, M., Rambone, G., Desideri, P., Carafa, M., Murtas, E., Riccieria, F. M., and Alhaiquea, F. (1998) J. Control Release 55, 57–66. [3] Morrison, N. A., Clark, R. C., Chen, Y. L., Talashek, T., and Sworn, G. (1999) Prog. Colloid Polym. Sci. 114, 127–131. [4] Bajaj, I., and Singhal, R. (2007) Food Chem. 104, 1472–1477. [5] Dennis, H. H., Thomas, O. M., and Paul, S. (1991) US Patent 4981520. [6] Vercaemer, C. J., Davies, S. N., Pafitis, D.G., Maintland, G. C., and Poyet, J. P. (1997) US Patent 5697441. [7] Plank, J. (2004) Appl. Microbiol. Biotechnol. 66, 1–9. [8] Allen, F. L., Best, G. H., and Linfroth, T. A. (1991) US Patent 5004506. [9] Kang, K. S., and Pettitt, D. J. (1993) In Industrial Gums: Polysaccharides and Their Derivatives (BeMiller, J. N., and Whistler, R. L., eds.). pp. 341–397, Academic Press, San Diego, CA. [10] Hagiwara, A., Imai, N., Doi, Y., Sano, M., Tamano, S., Omoto, T., Asai, I., Yasuhara, K., and Hayashi, S. (2010) J. Toxicol. Sci. 35, 493–501. [11] Paula, M. D., Goissis, G., and Martins, V. C. A. (2007) J. Mater. Sci. 18, 1683–1690.
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[12] Arockiasamy, S., and Banik, R. M. (2008) J. Biosci. Bioeng. 105, 204–210. [13] Suh, I. S., Herbst, H., Schumpe, A., and Deckwer, W. D. (1990) Biotechnol. Lett. 12, 201–206. [14] Amanullah, A., Satti, S., and Nienow, A. W. (1998) Biotechnol. Prog. 14, 265–269. [15] Li, H., Xu, H., Li, S., Feng, X. H., Xu, H., and Ouyang, P. K. (2011) Process Biochem. 46, 1172–1178. [16] Xu, H., Xu, X. Y., Dong, S. H., Li, S., and Liang, J. F. (2012) China Patent Application 201210504461.3. [17] Shang, F., Wen, S. H., Wang, X., and Tan, T. W. (2006) J. Biosci. Bioeng. 101, 38–41. [18] Xu, H., Jiang, M., Li, H., Lu, D. Q., and Ouyang, P. K. (2005) Process Biochem. 40, 519–523. [19] Banik, R. M., and Santhiagu, A. (2006) Biotechnol. Lett. 28, 1347–1350. [20] Wang, Z. J., Wang, H. Y., Li, Y. L., Chu, J., Huang, M. Z., Zhuang, Y. P., and Zhang, S. L. (2010) Bioresour. Technol. 101, 2845–2852. [21] Xiao, J., Shi, Z. P., Gao, P., Feng, H. J., Duan, Z. Y., and Mao, Z. G. (2006) Bioproc. Biosyst. Eng. 29, 109–117.
Rhamsan Gum Production by Sphingomonas sp. CGMCC 6833
