Bioprocess Biosyst Eng (2009) 32:755–763 DOI 10.1007/s00449-009-0300-6
ORIGINAL PAPER
Comparative study on the influence of dissolved oxygen control approaches on the microbial hyaluronic acid production of Streptococcus zooepidemicus Long Liu Æ Guocheng Du Æ Jian Chen Æ Miao Wang Æ Jun Sun
Received: 25 August 2008 / Accepted: 10 January 2009 / Published online: 11 February 2009 Ó Springer-Verlag 2009
Abstract Three different dissolved oxygen (DO) control approaches were proposed to improve hyaluronic acid (HA) production: a three-stage agitation speed control approach, a two-stage DO control approach, and an oxygen vector perfluorodecalin (PFC) applied approach. In the three-stage agitation speed control approach, agitation speed was 200 rpm during 0–8 h, 400 rpm during 8–12 h, and 600 rpm during 12–20 h. In the two-stage DO control strategy, DO was controlled at above 10% during 0–8 h and at 5% during 8–20 h. In the PFC applied approach, PFC (3% v/v) was added at 8 h. HA production reached 5.5 g/L in the three-stage agitation speed control culture model, and 6.3 g/L in two-stage DO control culture model, and 6.6 g/L in the PFC applied culture model. Compared with the other two DO control approaches, the PFC applied
L. Liu G. Du School of Biotechnology, Jiangnan University, 214122 Wuxi, China L. Liu G. Du (&) J. Chen Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, 214122 Wuxi, China e-mail:
[email protected] J. Chen (&) State Key Laboratory of Food Science and Technology, Jiangnan University, 214122 Wuxi, China e-mail:
[email protected] M. Wang School of Food Science and Technology, Jiangnan University, 214122 Wuxi, China J. Sun Institute of Information Technology, Jiangnan University, 214122 Wuxi, China
approach had a lower shear stress and thus a higher HA production was achieved. Keywords Hyaluronic acid Streptococcus zooepidemicus Oxygen mass transfer Two-stage Oxygen vector
Introduction Hyaluronic acid (HA) is a linear glycosaminoglycan polysaccharide composed of repeating disaccharide units of alternative D-glucuronic acid (GlcUA) and N-acetylglucosamine (GlcNAc) [1]. With the unique physico-chemical and biological properties, such as high water-holding capacity, viscoelasticity and biocompatibility, HA finds wide applications in biomedical, food, healthcare and cosmetic fields [2–6]. Conventionally HA was extracted from animal tissue like rooster combs, and now is increasingly produced via microbial fermentation with a lower production cost. HA is synthesized as an extracellular capsule by group A and C streptococci. HA from microbial fermentation would become the predominant source in the biomedical and cosmetic markets, and thus it was crucial to improve HA productivity to meet the increasing market demand [7]. Microbial HA production was limited by many factors, such as the high broth viscosity and low oxygen mass transfer efficiency [8], the carbon and energy competitions among cell growth, lactic acid production and HA synthesis [9], and the inhibition of lactic acid on cell growth and HA synthesis and so on [10]. There are many reports regarding the optimization of microbial HA production in the literature. Hasegawa et al. [8] applied the maxblend fermentor with better mixing performance and oxygen
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mass transfer efficiency to improve HA productivity. Kim et al. [11] selected a strain producing high molecular weight HA and optimized the culture conditions including the temperature, pH, agitation speed, and aeration rate and so on. Huang et al. [7] enhanced HA productivity with a repeated batch culture mode, and Kim et al. [12] developed a novel cell growth-based multi-stage agitation speed and temperature control approach to improve HA production. In the previous work, we studied the influence of culture modes on microbial HA production [13] and enhanced HA production by a two-stage fed-batch and batch culture strategy to redirect the carbon flux from cell growth to HA synthesis [14]. Though much work has been conducted on microbial HA production, there are few reports concerning the influence of dissolved oxygen (DO) control approaches on HA production, despite the fact that oxygen mass transfer played an important role in the microbial HA production. In this work, the influence of different DO control approaches on the microbial HA production was investigated. Three culture models with different DO control approaches were applied: a three-stage agitation speed control culture model, a two-stage DO control culture model, and an oxygen vector perfluorodecalin (PFC) applied culture model. Considering the significant influence of broth rheology on oxygen mass transfer, we firstly characterized the kinetics of broth rheology in microbial HA production. To the best of our knowledge, this is the first report concerning the impact of different DO control approaches on the microbial HA production. Furthermore, the culture models with different DO control strategies proposed in this work may be helpful to the microbial production of the other biopolymers.
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trace element solution (pH 7.2). Culture medium was sterilized at 121 °C for 15 min. Batch culture of S. zooepidemicus One loop of cells from a fresh slant was transferred to 50 mL seed culture medium and cultured on a rotary shaker at 200 rpm and 37 °C for 12 h. The seed culture was inoculated into a 7-L fermentor (Model KL-7L, K3T Ko Bio Tech, Korea) containing 4.0 L culture medium. Agitation was provided by three-six-bladed disk turbines (diameter ratio of turbine and fermentor was 1:2.5). The pH was automatically controlled at 7.0 by adding 5 mol/L NaOH solution and temperature was maintained at 37 °C. Otherwise mentioned, aeration rate and agitation speed was 0.5 vvm and 200 rpm, respectively. The batch culture with a constant agitation speed of 200 rpm was referred as the control in this work. Analytical methods Lactic acid and sucrose were analyzed with the method we previously described [10]. Cell concentration was measured from optical density (OD) of culture broth at 660 nm with a spectrophotometer (722s spectrophotometer, China). HA concentration was measured by the carbazole method based on uronic acid determination [15]. Oxygen mass transfer coefficient KLa was measured according to the dynamic method [16]. Simply, KLa can be calculated as the slope of the linear Eq. 1: lnð1 EÞ ¼ KL aðt t0 Þ where E is given as E¼
Materials and methods Microorganism and media Streptococcus zooepidemicus WSH-24 used in this study was isolated by our lab. Fresh slants were cultured at 37 °C for 12 h and were used for inoculation. Slant culture medium consisted of (g/L): brain heart infusion (BHI) (Difco, Detroit, MI 48232-7058, USA) 37, glucose 10, yeast extract (Angel Yeast Co., Ltd, Hubei, China) 10, agar powder 20. Seed culture medium consisted of (g/L): sucrose 20, yeast extract 20, MgSO47H2O 2.0, MnSO44H2O 0.1, KH2PO4 2.0, CaCO3 20 and 1 mL trace elements solution. The trace element solution consisted of (g/L): CaCl2 2.0, ZnCl2 0.046 and CuSO45H2O 0.019. Fermentation medium contained (g/L): yeast extract 25, sucrose 70, K2SO4 1.3, MgSO47H2O 2.0, Na2HPO412H2O 6.2, FeSO47H2O 0.005 and 2.5 mL
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ð1Þ
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where C* is the saturation concentration of DO, C0 is the initial concentration of DO at time t0 when a hydrodynamic steady-state has been reestablished upon commencement of aeration, and C is the DO concentration at any time t. It should be noted that the dynamic method was only suitable for KLa measurement at a high DO level. With the gained data at high DO levels, a model correlating KLa with agitation speed, aeration rate and broth viscosity was obtained (Eq. 5). KLa was calculated from the established model when DO level was low (during 8–18 h). The apparent viscosity of the fermentation broth was estimated with the power-law equation as [17]: g ¼ KðcÞn1
ð3Þ
where g is the apparent viscosity (Pa s), K is the consistency index (Pa sn), n is the flow behavior index, and c is the average shear rate (s-1), which depended on
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the rotational speed of the impeller, as follows (for Rushton turbine, k is 11.5): c ¼ kN
during 8–18 h. Figure 2b shows that KLa decreased from 98 h-1 at 0 h to 3 h-1 at 18 h, in accordance with the changes of DO level over time. In a given culture system, KLa can be correlated with the agitation speed, aeration rate and apparent viscosity. As shown in Eq. 5, a correlation model was obtained by regression analysis of the experimental data.
ð4Þ
Results Kinetic analysis of broth rheology and oxygen mass transfer characteristics
KL a ¼ 66:96
Figure 1 shows the kinetics of broth rheology during microbial HA production. Broth apparent viscosity and the consistency index k increased with culture time and reached the maximum value of 375 mPa s and 8.0 Pa sn at 18 h, respectively. The flow behavior index n decreased from 1.0 at 0 h to a minimum value of 0.19 at 18 h, indicating that the pseudoplasticity of the culture broth increased with the culture time. The linear relationship between shear rate and shear stress indicated that culture broth was a kind of Newtonian fluid at the initial phase of culture process. With the increase of culture time, the broth exhibited a typical pseudoplastic behavior and the shear thinning phenomenon was observed. Usually oxygen mass transfer rate is a crucial factor for bioprocess optimization and also is an important parameter for scale up [18–21]. Figure 2 shows DO and KLa as a function of culture time. Figure 2a indicates that DO level decreased from 100% at 0 h to 0–1% of air saturation
where N is the agitation speed (rev/s), VS is the superficial air velocity (m/s), and g is the apparent viscosity (Pa s). It was indicated that the agitation speed had the most significant influence on KLa, whereas the influence of apparent viscosity on KLa seemed to be small due to the small exponent. Ryu and Humphrey [22] examined the effect of apparent viscosity on the mass transfer coefficient KLa for P. chrysogenum broth and proposed a correlation model with an apparent viscosity exponent of -0.85. Yagi and Yoshida [23] proposed a dimensionless correlation for KLa in sparged agitated vessels on the basis of experimental data for oxygen desorption from two non-Newtonian fluids (sodium polyacrylate solution and carboxymethyl cellulose solution), and the apparent viscosity exponent was -0.4. Compared with these correlation models, the exponent of apparent viscosity was smaller, maybe due to the difference of the culture system.
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Fig. 1 Time profiles of broth rheology in batch culture of S. zooepidemicus (open square 0 h; asterisks 6 h; open circle 12 h)
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Fig. 2 Changes of dissolved oxygen (DO) level and oxygen transfer coefficient KLa over time during batch culture of S. zooepidemicus
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Effect of three-stage agitation speed control approach on microbial HA production
Effect of two-stage DO control approach on the microbial HA production
The microbial HA production by batch culture of S. zooepidemicus with a constant agitation speed of 200 rpm was conducted. As we previously described [13], cells entered the exponential growth phase after 2 h of lag phase and reached a maximum concentration of 13.4 g/L at 14 h. The sucrose concentration decreased from 70 g/L at 0 h to 1.2 g/L at 20 h. HA concentration reached a maximal value of 5.0 g/L at 16 h, while a maximum lactic acid concentration of 55 g/L was observed at the end of batch culture. As indicated in Fig. 2, DO level decreased to 0–1% of air saturation at 8 h and became a limiting factor for microbial HA production. It was expected to improve HA production via the enhancement of oxygen mass transfer and DO level during 8–20 h. As revealed in Eq. 5, agitation speed was the mostly important factor influencing KLa, and thus a three-stage agitation speed control approach was proposed to achieve the increase of oxygen mass transfer rate. In this culture model, agitation speed was 200 rpm during 0–8 h, 400 rpm during 8–12 h, and 600 rpm during 12–20 h. Figure 3 shows the influence of three-stage agitation speed control approach on oxygen mass transfer and microbial HA production. The average KLa and DO level during 8–20 h increased from 16 h-1 and 0.5% of the control to 35 h-1 and 8% respectively in the three-stage agitation speed control culture model. Due to the alleviation of DO limitation, HA increased from 5.0 g/L of the control to 5.5 g/L in the three-stage agitation speed control culture model. There was little effect of three-stage agitation speed control approach on the cell concentration. This was maybe due to the fact that, on one hand, the increased DO level was favorable for cell growth, while on the other hand, the increased agitation speed caused a stronger shear stress to the cells.
Huang et al. [24] found that DO played a role as a stimulant in HA synthesis and there existed a critical DO level of 5% of air saturation for HA synthesis. In the previous work [25], we studied the effects of DO level on the microbial HA production in chemostat culture of S. zooepidemicus. The results revealed that there existed a critical DO level of 5% and 10% of air saturation respectively for HA synthesis and cell growth, in accordance with what reported by Huang et al. [24]. It was reported that the synthesis HA capsule could protect cells from the damage of oxygenderived free radicals [26]. In other words, an appropriate DO level could stimulate the synthesis of HA, and when DO level was higher than the critical level of 5%, the stimulation effect was not so significant. The carbon and energy competitions among cell growth, lactic acid production and HA synthesis limited the overproduction of HA. As discussed above, the existence of different critical DO levels for cell growth and HA production made it feasible to redirect the carbon flux from cell growth to HA synthesis via a two-stage DO control strategy. In this strategy, DO level was controlled at higher than 10% during 0–8 h and at 5% during 8–20 h via the automatic control of agitation speed. Figure 4 shows the effects of two-stage DO control approach on the microbial HA production. Cell concentration decreased from 13.4 g/L of the control to 12.1 g/L, and HA production increased from 5.0 g/L of the control to 6.3 g/L in the proposed two-stage DO control culture model. With the improved DO level, the decrease of lactic acid concentration from 55 to 45 g/L was observed. The results showed that the proposed two-stage DO control strategy not only improved the oxygen mass transfer rate, but also alleviated the carbon and energy competitions among cell growth, lactic acid
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Fig. 3 Influence of three-stage agitation speed control approach on the oxygen mass transfer characteristics and microbial HA production
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production and HA synthesis, and thus achieved the enhancement of HA productivity from 0.0694 to 0.0984 g L-1 h-1.
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Fig. 4 Influence of two-stage DO control approach on the microbial HA production
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or the two-stage DO control culture model. And thus the negative effect of high shear stress on cell growth and HA synthesis was unable to avoid. Then a question was proposed: how to improve HA production by improving oxygen mass transfer and DO level at a low agitation speed (low shear stress)? Effect of oxygen vector PFC applied approach on microbial HA production A novel approach to achieve better oxygen supply is to increase the solubility of oxygen in the culture medium by the addition of oxygen vectors. This approach includes the application of oxygen vectors such as hemoglobin, hydrocarbons and perfluorocarbons [27]. The oxygen solubility in these compounds is about 15–20 times higher than that in water [28]. Perfluorocarbons (PFCs) are petroleum-based compounds synthesized by substituting fluorine for the hydrogen molecules of hydrocarbons. They are both stable and chemically inert due to the presence of very strong carbon-fluorine bonds. PFC is a derivative of decalin in which all of the hydrogen atoms are replaced by fluorine atoms. PFC is chemically and biologically inert, and has a super oxygen dissolving ability as high as 127.8 mg/L at 25 °C [29]. In this work, PFC was used as an oxygen vector to improve oxygen mass transfer in the second phase (8–20 h) of microbial HA production by batch culture of S. zooepidemicus. The PFC with different
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Fig. 5 Effect of oxygen vector PFC applied approach on the broth rheology and oxygen mass transfer characteristics
concentrations (1, 3, 5, 7% v/v) was added at 8 h in microbial HA production. It was found that the addition of PFC (3%) could improve the average DO level during 8–20 h to 5%, and the addition of PFC with a concentration higher than 3% could not further improve oxygen mass transfer and DO level due to the increased broth viscosity with the addition of PFC. Figure 5 shows the kinetics of broth rheology and oxygen mass transfer at 3% of PFC concentration. The consistency index k increased while the flow behavior index n decreased compared with the control without PFC addition, indicating that PFC addition had a significant influence on broth rheology of the culture broth. The average KLa during 8–20 h increased from 16 h-1 of the control to 30 h-1, and the average DO level during 8–20 h increased from 0.5% of the control to 5%. These results indicated that the PFC addition could significantly improve the oxygen mass transfer efficiency and DO level at a low agitation speed. Figure 6 shows the metabolic kinetics of microbial HA production at 3% of PFC concentration. Cell and HA concentration increased from 13.4 and 5.0 g/L of the control to 14.2 and 6.6 g/L, respectively. Due to the production of lactic acid was inhibited at a high DO level, lactic acid concentration decreased from 55 g/L of the control to 43 g/L at a PFC concentration of 3%. It should be noted that the inhibition of lactic acid on cell growth and HA synthesis was alleviated at a higher DO level achieved by the three-stage
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agitation speed control strategy, two-stage DO control strategy, or the addition of PFC. That is to say, the alleviation of lactic acid inhibition on HA synthesis also contributed to the enhancement of HA production with the proposed different DO control approaches.
Discussion HA is a commercially valuable medical biopolymer increasingly produced via microbial fermentation, and HA from microbial fermentation would become the predominant source in the biomedical and cosmetic markets [7]. Therefore, the microbial HA production is attracting more and more research interests. Huang et al. [7] investigated the production of HA with repeated batch culture, by which HA productivity was increased by 2.5-fold compared with the batch culture. Kim et al. [12] developed a novel cell growth based multi-stage agitation speed and temperature control approach to improve HA production, which achieved 5.4 g/L on an industrial scale. HA production increased from 5.5 g/L of the control to 7.5 g/L by expressing polyhydroxybutyrate synthesis genes in S. zooepidemicus to adjust the cellular oxidation/ reduction potential [30]. A maxblend fermentor with better mixing performance was applied to the microbial HA production and HA production was increased by 20% [8].
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In fact, one important factor limiting microbial HA production is the low oxygen mass transfer efficiency resulted from the high broth viscosity, and the improvement of oxygen mass transfer and DO level was considered as an effective means to enhance HA productivity. Therefore, developing an efficient DO control approach was very important to improve HA productivity. However, there are few reports regarding the influence of DO control approach on the microbial HA production. In this work, three different DO control approaches were proposed to improve the oxygen mass transfer efficiency to enhance HA productivity: three-stage agitation speed control approach, two-stage DO control approach, and the oxygen vector PFC applied approach. In the proposed three-stage agitation speed control approach, agitation speed was 200 rpm during 0–8 h, 400 rpm during 8–12 h, and 600 rpm during 12–20 h. The three-stage agitation speed control culture model had a higher oxygen mass transfer efficiency and DO level during 8–20 h compared with the control, in which the agitation speed was a constant of 200 rpm. HA concentration increased from 5.0 g/L of the control to 5.5 g/L in the three-stage agitation speed control culture model. A two-stage DO control approach was also applied to the microbial HA production. In this culture model, DO level was controlled higher than 10% during 0–8 h and at 5% during 8–20 h. There existed a critical DO level of 5 and 10% for HA synthesis and cell growth, respectively.
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The developed two-stage DO control approach was based on the fact that the alleviation of the carbon competition between cell growth and HA synthesis would be favorable for the enhanced HA productivity [9]. In this approach, not only the oxygen mass transfer efficiency during 8–20 h was improved, but also the carbon competition between cell growth and HA synthesis was alleviated. Therefore, the two-stage DO control culture model achieved a higher HA production compared with the three-stage agitation speed control approach. In the two-stage DO control approach, a high agitation speed was needed to maintain a high DO level during 8–20 h, and therefore the shear stress to the cell was unable to avoid. To improve the oxygen mass transfer efficiency at a low agitation speed, an oxygen vector PFC applied approach was applied to the microbial HA production. In this approach, the oxygen vector PFC was added at 8 h to improve the oxygen mass transfer efficiency and DO level during 8–20 h. The application of PFC as an oxygen vector can improve KLa and DO level significantly without increasing agitation speed, and thus the shear stress to the cells was able to avoid. Therefore, HA production increased from 5.0 g/L of the control to 6.6 g/L in the oxygen vector PFC applied culture model. The influence of different DO control approaches on the microbial HA production indicated that the application of oxygen vector was an efficient approach for the microbial HA production with the significant improvement of oxygen mass transfer efficiency and low shear stress. Conclusion This contribution compared the influence of different DO control approaches on the microbial HA production of S. zooepidemicus. Three DO control culture models were considered: three-stage agitation speed control approach, two-stage DO control culture model, and the oxygen vector PFC applied culture model. HA increased from 5.0 g/L of the control to 6.3 g/L in the two-stage DO control culture model, which not only improved oxygen mass transfer efficiency, but also alleviated the carbon competition between cell growth and HA synthesis. HA production increased from 5.0 g/L of the control to 6.6 g/L in the PFC applied culture model with a low shear stress as well as the significant improvement of oxygen mass transfer efficiency. The knowledge obtained here may be helpful to the other microbial biopolymer production. Acknowledgments This project was financially supported by Program for Changjiang Scholars and Innovative Research Team in University (No. IRT0532), the National Science Fund for Distinguished Young Scholars of China (No. 20625619), Program for Cultivation and Innovation of Graduate Students in Jiangsu Province (CX08B_128Z), and 973 Project (2007CB714306).
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