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Production of hyaluronic acid by repeated batch fermentation
Biochemical Engineering Journal 40 (2008) 460–464
Production of hyaluronic acid by repeated batch fermentation Wei-Chih Huang a , Shu-Jen Chen b , Teh-Liang Chen a,∗ a
b
Department of Chemical Engineering, National Cheng Kung University, Tainan 701, Taiwan Department of Chemical and Material Engineering, National Kaohsiung University of Applied Sciences, Kaohsiung 807, Taiwan Received 26 September 2007; received in revised form 9 December 2007; accepted 11 January 2008
Abstract The production of hyaluronic acid (HA) by Streptococcus zooepidemicus with repeated batch fermentation has been investigated. It was found that, with conventional operation, both maximal specific growth rate (μm ) and specific HA productivity (YP/X ) decreased with increasing seed volume, suggesting that there exist some inhibitors in the broth. The removal of liquid in the seed was first attempted by installing nonwoven fabrics (NWF) in the fermentor to retain some of the cells when draining the broth. However, this resulted in a loss of HA productivity, which in turn was attributed to the growth of a sticky, non-HA-producing mutant on the NWF. Using an external cartridge filter to partially retain the cells, followed by back-washing the filter with fresh medium for seeding, μm and YP/X could be maintained successfully at their batch levels during the repeated cycles. In an operation that seeded 31% cell, the volumetric production rate of the repeated batch culture (0.59 g HA L−1 h−1 ) was found to be 2.5-fold of the batch culture (0.24 g HA L−1 h−1 ). © 2008 Elsevier B.V. All rights reserved. Keywords: Fermentation; Batch processing; Submerged culture; Biosynthesis; Hyaluronic acid; Streptococcus zooepidemicus
1. Introduction Hyaluronic acid (HA) is a mucopolysaccharide consisting of alternating N-acetyl-d-glucosamine and d-glucuronic acid, which has received great interest in the medical and cosmetic markets [1–3]. In recent years, HA from microbial fermentation, rather than extraction from animal sources, is receiving increased attention for avoidance of cross-species viral infection. HA fermentations have been mostly by Streptococci spp., where HA is a capsular biopolymer shedding to the medium [4]. To aid the competence of the fermentation process, the development of an economical process for mass production is necessary. In the literature, most reports on HA fermentation have been based on batch culture [5–9]. A major drawback of batch culture is a long turnaround time, which greatly decreases the volumetric production rate, and this in turn results in a high fixed cost per unit product. There are two approaches that could be employed to increase the volumetric production rate. One is to increase HA concentration in the fermentor, which can be accomplished either through
∗
Corresponding author. Tel.: +886 6 2757575x62660; fax: +886 6 2344496. E-mail address: [email protected] (T.-L. Chen).
1369-703X/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.bej.2008.01.021
high-cell-density fermentation or through a high-yield strain. However, we found that, as the HA concentration higher than 4 g/L, the broth became too viscous to achieve efficient agitation and aeration; as a result, the benefit of high HA concentration is worn down by the defect of low efficiency of HA synthesis. The other approach is to skip the turnaround phase by using continuous culture. Continuous culture could also offer two benefits. First, cell growth can be maintained at the exponential phase, so that the excretion of cell wall proteins at the stationary phase [4] can be avoided. Second, the cells can be controlled to grow at a lower specific growth rate, which might result in HA of higher molecular weight [5]. However, continuous culture has its inherent defect—low efficiency of substrate utilization; and this is the primary reason why continuous culture is seldom used in a commercial process. Another adverse factor concerned is that the efficiency of HA production would decrease during prolonged operation [10,11]. Accordingly, repeated batch culture seems a promising mode for HA fermentation, because it skips the turnaround time and the lag phase. Unfortunately, no report on such subject has been published to date. The aim of this work was to explore the problems encountered during HA production by repeated batch culture, to rationalize the problems, and to propose a feasible operation strategy.
W.-C. Huang et al. / Biochemical Engineering Journal 40 (2008) 460–464
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2. Materials and methods S. zooepidemicus ATCC 39920 was used as the HA producer. The cells were maintained at −30 ◦ C in 50% (v/v) glycerol. The isolation of a pure culture was achieved by streaking onto TSB agar plates, which contained (per liter) 17 g of tryptone, 3 g of soytone, 2.5 g of glucose, 2.5 g of K2 HPO4 , 5 g of NaCl, and 15 g of agar. Precultures were prepared in a 500-mL shaking flask, with 100 mL of the TSB medium, at 37 ◦ C for 12 h. HA production was performed in a 3 L fermentor (MDL300, B. E. Marubishi, Japan), with a working volume of 2 L. The medium comprised of (per liter) 20 g of glucose, 10 g of yeast extract, 1.7 g of tryptone, 0.3 g of soytone, 2 g of NaCl, 2.5 g of K2 HPO4 and 1.5 g of MgSO4 ·7H2 O. The fermentor was inoculated with 5% (v/v) preculture, and operated at 37 ◦ C and pH 7.0 (with 5N NaOH). The aeration rate was 1 vvm, and the concentration of dissolved oxygen (DO) was controlled above 10% air saturation by mixing the inlet air with oxygen when necessary. Agitation was achieved with a gate impeller similar to that of Hiruta et al. [12], and an agitator speed of 400 rpm was used. In repeated batch culture, the end of each cycle was determined when there was a rise in DO level; it is a sign that the cells cease to grow. The medium concentration was adjusted according to the amount of cell in the seed, such that the repeated cycles were supposedly to achieve the same cell concentration as the batch level. The nonwoven fabric (NWF) used to retain the cells consists of a polypropylene core and a polyethylene surface. The fabric has a unit weight of 60 g/m2 and a specific gravity of 0.90. To equip the NWF in the fermentor, three pieces of the NWF (4 cm × 17 cm) were tied around the three baffles, unless otherwise noted. The cartridge filter used to retain the cells outside the fermentor had a diameter of 3 cm and a length of 30 cm, where the NWF was used as the filter medium. After draining the broth, the retained cells were released by back-washing with fresh medium to seed the fermentor. The back-wash procedure was ended with air-purge to avoid microbial growth in the cartridge. The amount of cells in the seed was adjusted by the amount of the NWF inside the filter as well as the number of the cartridges. Cell concentration was measured from optical density (OD) of the broth at 660 nm using a spectrophotometer. Owing to a change in cell morphology after entry into the stationary phase [13], the correlations of OD with dry cell weight (DCW) were DCW (g/L) = 0.399 × OD − 0.003 for the exponential growth phase, and DCW (g/L) = 0.456 × OD − 0.012 for the stationary phase. HA concentration was determined by the carbazole method [14], in which the optical density was measured at 525 nm and d-glucuronic acid was used as the standard.
Fig. 1. HA production by repeated batch culture, using 5% broth as the seed. Symbols: (䊉) cell density and () HA concentration.
estimated to be 0.59 h−1 ; and the specific productivity (YP/X ), defined as the ratio of total increment of HA to total increment of cell mass, was 0.72 g HA/g cell. μm together with YP/X are good indicators for assessing the efficiency of the repeated batch culture. The repeated batch culture was first performed using a seed of 5% broth. As can be seen in Fig. 1, both cell and HA concentrations could be maintained as in the batch culture for at least seven repeated cycles. However, μm and YP/X are reduced to 0.51 h−1 and 0.62 g HA/g cell, respectively. To increase the volumetric production rate, volumes of the seed were further increased to 10, 20, 30 and 40% of the broth, respectively. Fig. 2 depicts the profiles of using 40% broth as the seed. Unfortunately, although cell concentration could be maintained, HA production was reduced gradually to 1.3 g/L. The influence of seed volume in the repeated batch culture is summarized in Fig. 3, which shows both μm and YP/X decreased with increasing seed volume. It thus suggests that in the broth there exist some inhibitors that hinder cell growth and HA synthesis. It might also be possible that the inhibitors hinder cell growth, and a smaller specific growth rate results in a smaller specific HA productivity; in other words, the inhibitors might not function directly on HA synthesis. We therefore performed a fed-batch culture that was operated at a specific growth rate of 0.30 h−1 ,
3. Results and discussion The preliminary results of batch culture showed that the cells entered the stationary phase at 9 h, with a cell concentration of 3.2 g/L and a HA concentration of 2.3 g/L; in addition, the formation of HA was a growth-associated matter (data can also be seen in Fig. 1). The maximal specific growth rate (μm ) was
Fig. 2. HA production by repeated batch culture, using 40% broth as the seed. Symbols: (䊉) cell density and () HA concentration.
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Fig. 5. Comparison of batch fermentations between the non-mucoid mutant (䊉, ) and the normal strain (, ). Symbols: (䊉, ) cell density and (, ) HA concentration.
Fig. 3. Variations of μm and YP/X during the repeated batch culture, here showing μm and YP/X decrease with increasing volume of seed. The repeated cycle of 0 denotes the initial batch culture. Seed volume: (䊉) 5%, () 10%, () 20%, () 30%, and () 40% (v/v).
the lowest rate found in Fig. 3. However, the resulted YP/X was still 0.72 g HA/g cell (fermentation profiles not shown). This assumes that the inhibitors function on both cell growth and HA synthesis. With the assumption about the inhibitors, it is likely that both μm and YP/X can be maintained during the repeated cycles if the seed contains no liquid. We then installed NWF in the fermentor to retain some of the cells when draining the broth. The retained cells were then released and served as the seed for the next cycle. The fermentation profiles thus obtained are shown in Fig. 4, where the seed was estimated to be equivalent to a 3% inoculum. It can be seen that cell concentration (of the broth) could be maintained for two repeated cycles; thereafter, it decreased gradually. The decrease in cell concentration was found to be attributed to that more and more cells grew on the NWF. It
Fig. 4. HA production by repeated batch culture with NWF equipped in the fermentor. Symbols: (䊉) cell density and () HA concentration.
is worth noting that the NWF functioned merely to block the cells from draining in the first three cycles, yet surface growth occurred in the succeeding cycles. On the other hand, although the HA level could also be maintained for two repeated cycles, it decreased sharply after the third cycle, and eventually, HA production ceased at the sixth cycle. Nevertheless, the cells still grew at a μm of 0.60 h−1 throughout the repeated cycles. From Fig. 4, an important clue to the HA fermentation can be obtained: the cells grown on the NWF, and their descendants, must be a non-HA-producing mutant. To confirm this suspicion, cells on the NWF were isolated on an agar plate; and as expected, they appeared as small, rough colonies (diameter of 0.5–0.8 mm, non-mucoid) rather than large, smooth colonies (diameter of 3 mm, mucoid, normal strain) [10]. The percentages of the mutant in the broth at the end of each cycle were found to be 0, 0, 0, 0, 33, 79 and 100% for repeated cycles of 0 (the initial batch culture), 1, 2, 3, 4, 5 and 6, respectively. The tendency of increase in this percentage was in accordance with the reduction in the HA production. It is interesting to note that the mutant appeared after 32 h (the third repeated cycle), not from the beginning. This is similar to a previous report [10], where showed the occurrence of non-HA-producing cells was beyond 27 h in a chemostat culture. Furthermore, a comparison between the non-mucoid mutant and the normal strain was performed with batch fermentation, as shown in Fig. 5. It can be seen that the extent of cell growth as well as μm of both strains were quite similar, though the mutant had a shorter lag phase; nevertheless, the mutant showed no ability of HA production. Once again, the results of Fig. 5 are in agreement with the phenomena found in Fig. 4. It is worth noting that the microscopic observation of both strains showed the same cell morphology—they appeared as strands of 6–8 cells during the exponential growth phase. A similar result was also reported for group A streptococci [15]. To avoid growth of the non-mucoid mutant on the NWF, we tied the NWF to the agitator, instead of the baffles. However, similar results as in Fig. 4 were obtained (data not shown); the
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Fig. 6. HA production by repeated batch culture with a cartridge filter, where the amount of cell seeded was 3%. Symbols: (䊉) cell density and () HA concentration.
mutant is so sticky that it can cling onto the NWF, irrespective of the higher shear force. The above observations, together with previous works [6,10,13,16], suggest the following hypothesis for HA fermentation. S. zooepidemicus is an aerotolerant anaerobe; aeration has no effect on cell growth, but it markedly enhances HA production. During the exponential growth phase, the cocoid cells associate into strand by capsule for themselves to be shielded from oxygen. After entry into the stationary phase, some inhibitors that hinder cell growth and HA formation would appear in the broth, and this limits the repeated batch culture to using a seed of small volume. In continuous fermentations, a sticky non-mucoid mutant, which has the advantage of a longer retention time, would eventually become the prevailing species, and thus HA production ceases. The use of NWF, which provides a tremendous surface area for the sticky cells to cling onto, is an enticement for the prevalence of the non-HA-producing cells. According to the hypothesis, HA production by repeated batch culture can be performed as follows: partially block the cells with an external filter when draining the broth, followed by back-washing the filter with fresh medium to seed the fermentor. Fig. 6 shows the repeated batch culture with a cartridge filter, where the amount of cell seeded was estimated to be 3%. It can be seen that during the repeated cycles, not only cell and HA concentrations but also μm and YP/X could be maintained at their batch levels. Similar results were also obtained when seeding larger amounts of cell, as shown in Fig. 7; the strategy to operate the repeated batch culture was thus quite successful. It is perhaps worth mentioning that the external filter has an added benefit—it helps to remove the sticky, non-HA-producing mutant from the seed, if they would occur. As mentioned above, increasing the volumetric production rate, rather than increasing the product concentration, is the focus of HA fermentation. Fig. 8 shows the comparison of HA productivities in batch culture and in repeated batch cultures. The batch culture had a HA productivity of 0.24 g HA L−1 h−1 , not counting the turnaround time. With conventional repeated batch culture (seed containing liquid), the productivity would not increase with increasing seed volume; the beneficial effect of using large seed volume is worn down owing to the exis-
Fig. 7. Variations of μm and YP/X during the repeated batch culture, here showing μm and YP/X can be maintained at their batch levels if the seed contains no liquid. Amount of cell seeded: (䊉) 3%, () 6.7%, () 16%, and () 31%.
tence of inhibitors in the broth. On the contrary, if we seed the fermentor with cells only, the beneficial effect of high seeding ratio on the volumetric productivity can be manifest. As can be seen in Fig. 8, using a seed of 31% cell, a productivity of 0.59 g HA L−1 h−1 could be obtained, which was 2.5-fold of the batch culture.
Fig. 8. Comparison of volumetric productivities of HA between batch culture (repeated cycle of 0) and repeated batch culture. In the upper panel (seed containing liquid), the seed volumes were 5% (䊉), 10% (), 20% (), 30% (), and 40% (). In the lower panel (seed containing no liquid), the amounts of cell inoculated were 3% (䊉), 6.7% (), 16% (), and 31% ().
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4. Conclusions HA from microbial fermentation would become the predominant source in the medical and cosmetic markets. The improvement in volumetric productivity is crucial to HA fermentation, and repeated batch culture seems to be the most feasible approach. The problem encountered in conventional repeated batch fermentation of HA by S. zooepidemicus is the existence of some inhibitors in the broth, which wears down the benefit of using large seed volume. Equipping a filtration medium (NWF, in this work) inside the fermentor to retain the cells for serving as the seed would lead to a more serious problem: a sticky, nonHA-producing strain would grow on the solid surface and soon becomes the prevailing species. With an external cartridge filter to retain the cells when draining the broth, the repeated batch culture can be employed successfully for HA production. Acknowledgement This study was supported by research grant NSC96-2221-E006-271 provided by the National Science Council of Taiwan. References [1] I.F. Radaeva, G.A. Kostina, A.V. Zmievskii, Hyaluronic acid: biological role, structure, synthesis, isolation, purification, and applications, Appl. Biochem. Microbiol. 33 (1997) 111–115. [2] L. Lapˇcik Jr., L. Lapˇcik, S. De Smedt, J. Demeester, P. Chabreˇcek, Hyaluronan: preparation, structure, properties, and applications, Chem. Rev. 8 (1998) 2663–2684. [3] B. Fong Chong, L.M. Blank, R. Mclaughlin, L.K. Nielsen, Microbial hyaluronic acid production, Appl. Microbiol. Biotechnol. 66 (2005) 341–351.
[4] A. Mausolf, J. Jungmann, H. Robenek, P. Prehm, Shedding of hyaluronate synthase from streptococci, Biochem. J. 267 (1990) 191–196. [5] D.C. Armstrong, M.R. Johns, Culture conditions affect the molecular weight properties of hyaluronic acid produced by Streptococcus zooepidemicus, Appl. Environ. Microbiol. 63 (1997) 2759–2764. [6] W.C. Huang, S.J. Chen, T.L. Chen, The role of dissolved oxygen and function of agitation in hyaluronic acid fermentation, Biochem. Eng. J. 32 (2006) 239–243. [7] M.R. Johns, L.T. Goh, A. Oeggerli, Effect of pH, agitation and aeration on hyaluronic acid production by Streptococcus zooepidemicus, Biotechnol. Lett. 16 (1994) 507–512. [8] J.H. Kim, S.J. Yoo, D.K. Oh, Y.G. Kweon, D.W. Park, C.H. Lee, G.H. Gil, Selection of a Streptococcus equi mutant and optimization of culture conditions for the production of high molecular weight hyaluronic acid, Enzyme Microb. Technol. 19 (1996) 440–445. [9] I. van de Rijn, R.E. Kessler, Growth characteristics of group A streptococci in a new chemically defined medium, Infect. Immun. 27 (1980) 444–448. [10] L.M. Blank, R.L. McLaughlin, L.K. Nielsen, Stable production of hyaluronic acid in Streptococcus zooepidemicus chemostats operated at high dilution rate, Biotechnol. Bioeng. 90 (2005) 685–693. [11] D.C. Ellwood, C.G.T. Evans, G.M. Dunn, N. McInnes, R.G. Yeo, K.J. Smith, Production of hyaluronic acid, US Patent 5,411,874 (1995). [12] O. Hiruta, K. Yamamura, H. Takebe, T. Futamura, K. Iinuma, H. Tanaka, Application of Maxblend fermentor for microbial process, J. Ferment. Bioeng. 83 (1997) 79–86. [13] W.C. Huang, S.J. Chen, T.L. Chen, Modeling the microbial production of hyaluronic acid, J. Chin. Inst. Chem. Engrs. 38 (2007) 355–359. [14] T. Bitter, H.M. Muir, A modified uronic acid carbazole reaction, Anal. Biochem. 4 (1962) 330–334. [15] B.A.B. Leonard, M. Woischnik, A. Podbielski, Production of stabilized virulence factor-negative variants by group A streptococci during stationary phase, Infect. Immun. 66 (1998) 3841–3847. [16] P.P. Cleary, A. Larkin, Hyaluronic acid capsule: strategy for oxygen resistance in group A Streptococci, J. Bacteriol. 140 (1979) 1090–1097.
Production of hyaluronic acid by repeated batch fermentation Wei-Chih Huang a , Shu-Jen Chen b , Teh-Liang Chen a,∗ a
b
Department of Chemical Engineering, National Cheng Kung University, Tainan 701, Taiwan Department of Chemical and Material Engineering, National Kaohsiung University of Applied Sciences, Kaohsiung 807, Taiwan Received 26 September 2007; received in revised form 9 December 2007; accepted 11 January 2008
Abstract The production of hyaluronic acid (HA) by Streptococcus zooepidemicus with repeated batch fermentation has been investigated. It was found that, with conventional operation, both maximal specific growth rate (μm ) and specific HA productivity (YP/X ) decreased with increasing seed volume, suggesting that there exist some inhibitors in the broth. The removal of liquid in the seed was first attempted by installing nonwoven fabrics (NWF) in the fermentor to retain some of the cells when draining the broth. However, this resulted in a loss of HA productivity, which in turn was attributed to the growth of a sticky, non-HA-producing mutant on the NWF. Using an external cartridge filter to partially retain the cells, followed by back-washing the filter with fresh medium for seeding, μm and YP/X could be maintained successfully at their batch levels during the repeated cycles. In an operation that seeded 31% cell, the volumetric production rate of the repeated batch culture (0.59 g HA L−1 h−1 ) was found to be 2.5-fold of the batch culture (0.24 g HA L−1 h−1 ). © 2008 Elsevier B.V. All rights reserved. Keywords: Fermentation; Batch processing; Submerged culture; Biosynthesis; Hyaluronic acid; Streptococcus zooepidemicus
1. Introduction Hyaluronic acid (HA) is a mucopolysaccharide consisting of alternating N-acetyl-d-glucosamine and d-glucuronic acid, which has received great interest in the medical and cosmetic markets [1–3]. In recent years, HA from microbial fermentation, rather than extraction from animal sources, is receiving increased attention for avoidance of cross-species viral infection. HA fermentations have been mostly by Streptococci spp., where HA is a capsular biopolymer shedding to the medium [4]. To aid the competence of the fermentation process, the development of an economical process for mass production is necessary. In the literature, most reports on HA fermentation have been based on batch culture [5–9]. A major drawback of batch culture is a long turnaround time, which greatly decreases the volumetric production rate, and this in turn results in a high fixed cost per unit product. There are two approaches that could be employed to increase the volumetric production rate. One is to increase HA concentration in the fermentor, which can be accomplished either through
∗
Corresponding author. Tel.: +886 6 2757575x62660; fax: +886 6 2344496. E-mail address: [email protected] (T.-L. Chen).
1369-703X/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.bej.2008.01.021
high-cell-density fermentation or through a high-yield strain. However, we found that, as the HA concentration higher than 4 g/L, the broth became too viscous to achieve efficient agitation and aeration; as a result, the benefit of high HA concentration is worn down by the defect of low efficiency of HA synthesis. The other approach is to skip the turnaround phase by using continuous culture. Continuous culture could also offer two benefits. First, cell growth can be maintained at the exponential phase, so that the excretion of cell wall proteins at the stationary phase [4] can be avoided. Second, the cells can be controlled to grow at a lower specific growth rate, which might result in HA of higher molecular weight [5]. However, continuous culture has its inherent defect—low efficiency of substrate utilization; and this is the primary reason why continuous culture is seldom used in a commercial process. Another adverse factor concerned is that the efficiency of HA production would decrease during prolonged operation [10,11]. Accordingly, repeated batch culture seems a promising mode for HA fermentation, because it skips the turnaround time and the lag phase. Unfortunately, no report on such subject has been published to date. The aim of this work was to explore the problems encountered during HA production by repeated batch culture, to rationalize the problems, and to propose a feasible operation strategy.
W.-C. Huang et al. / Biochemical Engineering Journal 40 (2008) 460–464
461
2. Materials and methods S. zooepidemicus ATCC 39920 was used as the HA producer. The cells were maintained at −30 ◦ C in 50% (v/v) glycerol. The isolation of a pure culture was achieved by streaking onto TSB agar plates, which contained (per liter) 17 g of tryptone, 3 g of soytone, 2.5 g of glucose, 2.5 g of K2 HPO4 , 5 g of NaCl, and 15 g of agar. Precultures were prepared in a 500-mL shaking flask, with 100 mL of the TSB medium, at 37 ◦ C for 12 h. HA production was performed in a 3 L fermentor (MDL300, B. E. Marubishi, Japan), with a working volume of 2 L. The medium comprised of (per liter) 20 g of glucose, 10 g of yeast extract, 1.7 g of tryptone, 0.3 g of soytone, 2 g of NaCl, 2.5 g of K2 HPO4 and 1.5 g of MgSO4 ·7H2 O. The fermentor was inoculated with 5% (v/v) preculture, and operated at 37 ◦ C and pH 7.0 (with 5N NaOH). The aeration rate was 1 vvm, and the concentration of dissolved oxygen (DO) was controlled above 10% air saturation by mixing the inlet air with oxygen when necessary. Agitation was achieved with a gate impeller similar to that of Hiruta et al. [12], and an agitator speed of 400 rpm was used. In repeated batch culture, the end of each cycle was determined when there was a rise in DO level; it is a sign that the cells cease to grow. The medium concentration was adjusted according to the amount of cell in the seed, such that the repeated cycles were supposedly to achieve the same cell concentration as the batch level. The nonwoven fabric (NWF) used to retain the cells consists of a polypropylene core and a polyethylene surface. The fabric has a unit weight of 60 g/m2 and a specific gravity of 0.90. To equip the NWF in the fermentor, three pieces of the NWF (4 cm × 17 cm) were tied around the three baffles, unless otherwise noted. The cartridge filter used to retain the cells outside the fermentor had a diameter of 3 cm and a length of 30 cm, where the NWF was used as the filter medium. After draining the broth, the retained cells were released by back-washing with fresh medium to seed the fermentor. The back-wash procedure was ended with air-purge to avoid microbial growth in the cartridge. The amount of cells in the seed was adjusted by the amount of the NWF inside the filter as well as the number of the cartridges. Cell concentration was measured from optical density (OD) of the broth at 660 nm using a spectrophotometer. Owing to a change in cell morphology after entry into the stationary phase [13], the correlations of OD with dry cell weight (DCW) were DCW (g/L) = 0.399 × OD − 0.003 for the exponential growth phase, and DCW (g/L) = 0.456 × OD − 0.012 for the stationary phase. HA concentration was determined by the carbazole method [14], in which the optical density was measured at 525 nm and d-glucuronic acid was used as the standard.
Fig. 1. HA production by repeated batch culture, using 5% broth as the seed. Symbols: (䊉) cell density and () HA concentration.
estimated to be 0.59 h−1 ; and the specific productivity (YP/X ), defined as the ratio of total increment of HA to total increment of cell mass, was 0.72 g HA/g cell. μm together with YP/X are good indicators for assessing the efficiency of the repeated batch culture. The repeated batch culture was first performed using a seed of 5% broth. As can be seen in Fig. 1, both cell and HA concentrations could be maintained as in the batch culture for at least seven repeated cycles. However, μm and YP/X are reduced to 0.51 h−1 and 0.62 g HA/g cell, respectively. To increase the volumetric production rate, volumes of the seed were further increased to 10, 20, 30 and 40% of the broth, respectively. Fig. 2 depicts the profiles of using 40% broth as the seed. Unfortunately, although cell concentration could be maintained, HA production was reduced gradually to 1.3 g/L. The influence of seed volume in the repeated batch culture is summarized in Fig. 3, which shows both μm and YP/X decreased with increasing seed volume. It thus suggests that in the broth there exist some inhibitors that hinder cell growth and HA synthesis. It might also be possible that the inhibitors hinder cell growth, and a smaller specific growth rate results in a smaller specific HA productivity; in other words, the inhibitors might not function directly on HA synthesis. We therefore performed a fed-batch culture that was operated at a specific growth rate of 0.30 h−1 ,
3. Results and discussion The preliminary results of batch culture showed that the cells entered the stationary phase at 9 h, with a cell concentration of 3.2 g/L and a HA concentration of 2.3 g/L; in addition, the formation of HA was a growth-associated matter (data can also be seen in Fig. 1). The maximal specific growth rate (μm ) was
Fig. 2. HA production by repeated batch culture, using 40% broth as the seed. Symbols: (䊉) cell density and () HA concentration.
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Fig. 5. Comparison of batch fermentations between the non-mucoid mutant (䊉, ) and the normal strain (, ). Symbols: (䊉, ) cell density and (, ) HA concentration.
Fig. 3. Variations of μm and YP/X during the repeated batch culture, here showing μm and YP/X decrease with increasing volume of seed. The repeated cycle of 0 denotes the initial batch culture. Seed volume: (䊉) 5%, () 10%, () 20%, () 30%, and () 40% (v/v).
the lowest rate found in Fig. 3. However, the resulted YP/X was still 0.72 g HA/g cell (fermentation profiles not shown). This assumes that the inhibitors function on both cell growth and HA synthesis. With the assumption about the inhibitors, it is likely that both μm and YP/X can be maintained during the repeated cycles if the seed contains no liquid. We then installed NWF in the fermentor to retain some of the cells when draining the broth. The retained cells were then released and served as the seed for the next cycle. The fermentation profiles thus obtained are shown in Fig. 4, where the seed was estimated to be equivalent to a 3% inoculum. It can be seen that cell concentration (of the broth) could be maintained for two repeated cycles; thereafter, it decreased gradually. The decrease in cell concentration was found to be attributed to that more and more cells grew on the NWF. It
Fig. 4. HA production by repeated batch culture with NWF equipped in the fermentor. Symbols: (䊉) cell density and () HA concentration.
is worth noting that the NWF functioned merely to block the cells from draining in the first three cycles, yet surface growth occurred in the succeeding cycles. On the other hand, although the HA level could also be maintained for two repeated cycles, it decreased sharply after the third cycle, and eventually, HA production ceased at the sixth cycle. Nevertheless, the cells still grew at a μm of 0.60 h−1 throughout the repeated cycles. From Fig. 4, an important clue to the HA fermentation can be obtained: the cells grown on the NWF, and their descendants, must be a non-HA-producing mutant. To confirm this suspicion, cells on the NWF were isolated on an agar plate; and as expected, they appeared as small, rough colonies (diameter of 0.5–0.8 mm, non-mucoid) rather than large, smooth colonies (diameter of 3 mm, mucoid, normal strain) [10]. The percentages of the mutant in the broth at the end of each cycle were found to be 0, 0, 0, 0, 33, 79 and 100% for repeated cycles of 0 (the initial batch culture), 1, 2, 3, 4, 5 and 6, respectively. The tendency of increase in this percentage was in accordance with the reduction in the HA production. It is interesting to note that the mutant appeared after 32 h (the third repeated cycle), not from the beginning. This is similar to a previous report [10], where showed the occurrence of non-HA-producing cells was beyond 27 h in a chemostat culture. Furthermore, a comparison between the non-mucoid mutant and the normal strain was performed with batch fermentation, as shown in Fig. 5. It can be seen that the extent of cell growth as well as μm of both strains were quite similar, though the mutant had a shorter lag phase; nevertheless, the mutant showed no ability of HA production. Once again, the results of Fig. 5 are in agreement with the phenomena found in Fig. 4. It is worth noting that the microscopic observation of both strains showed the same cell morphology—they appeared as strands of 6–8 cells during the exponential growth phase. A similar result was also reported for group A streptococci [15]. To avoid growth of the non-mucoid mutant on the NWF, we tied the NWF to the agitator, instead of the baffles. However, similar results as in Fig. 4 were obtained (data not shown); the
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Fig. 6. HA production by repeated batch culture with a cartridge filter, where the amount of cell seeded was 3%. Symbols: (䊉) cell density and () HA concentration.
mutant is so sticky that it can cling onto the NWF, irrespective of the higher shear force. The above observations, together with previous works [6,10,13,16], suggest the following hypothesis for HA fermentation. S. zooepidemicus is an aerotolerant anaerobe; aeration has no effect on cell growth, but it markedly enhances HA production. During the exponential growth phase, the cocoid cells associate into strand by capsule for themselves to be shielded from oxygen. After entry into the stationary phase, some inhibitors that hinder cell growth and HA formation would appear in the broth, and this limits the repeated batch culture to using a seed of small volume. In continuous fermentations, a sticky non-mucoid mutant, which has the advantage of a longer retention time, would eventually become the prevailing species, and thus HA production ceases. The use of NWF, which provides a tremendous surface area for the sticky cells to cling onto, is an enticement for the prevalence of the non-HA-producing cells. According to the hypothesis, HA production by repeated batch culture can be performed as follows: partially block the cells with an external filter when draining the broth, followed by back-washing the filter with fresh medium to seed the fermentor. Fig. 6 shows the repeated batch culture with a cartridge filter, where the amount of cell seeded was estimated to be 3%. It can be seen that during the repeated cycles, not only cell and HA concentrations but also μm and YP/X could be maintained at their batch levels. Similar results were also obtained when seeding larger amounts of cell, as shown in Fig. 7; the strategy to operate the repeated batch culture was thus quite successful. It is perhaps worth mentioning that the external filter has an added benefit—it helps to remove the sticky, non-HA-producing mutant from the seed, if they would occur. As mentioned above, increasing the volumetric production rate, rather than increasing the product concentration, is the focus of HA fermentation. Fig. 8 shows the comparison of HA productivities in batch culture and in repeated batch cultures. The batch culture had a HA productivity of 0.24 g HA L−1 h−1 , not counting the turnaround time. With conventional repeated batch culture (seed containing liquid), the productivity would not increase with increasing seed volume; the beneficial effect of using large seed volume is worn down owing to the exis-
Fig. 7. Variations of μm and YP/X during the repeated batch culture, here showing μm and YP/X can be maintained at their batch levels if the seed contains no liquid. Amount of cell seeded: (䊉) 3%, () 6.7%, () 16%, and () 31%.
tence of inhibitors in the broth. On the contrary, if we seed the fermentor with cells only, the beneficial effect of high seeding ratio on the volumetric productivity can be manifest. As can be seen in Fig. 8, using a seed of 31% cell, a productivity of 0.59 g HA L−1 h−1 could be obtained, which was 2.5-fold of the batch culture.
Fig. 8. Comparison of volumetric productivities of HA between batch culture (repeated cycle of 0) and repeated batch culture. In the upper panel (seed containing liquid), the seed volumes were 5% (䊉), 10% (), 20% (), 30% (), and 40% (). In the lower panel (seed containing no liquid), the amounts of cell inoculated were 3% (䊉), 6.7% (), 16% (), and 31% ().
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4. Conclusions HA from microbial fermentation would become the predominant source in the medical and cosmetic markets. The improvement in volumetric productivity is crucial to HA fermentation, and repeated batch culture seems to be the most feasible approach. The problem encountered in conventional repeated batch fermentation of HA by S. zooepidemicus is the existence of some inhibitors in the broth, which wears down the benefit of using large seed volume. Equipping a filtration medium (NWF, in this work) inside the fermentor to retain the cells for serving as the seed would lead to a more serious problem: a sticky, nonHA-producing strain would grow on the solid surface and soon becomes the prevailing species. With an external cartridge filter to retain the cells when draining the broth, the repeated batch culture can be employed successfully for HA production. Acknowledgement This study was supported by research grant NSC96-2221-E006-271 provided by the National Science Council of Taiwan. References [1] I.F. Radaeva, G.A. Kostina, A.V. Zmievskii, Hyaluronic acid: biological role, structure, synthesis, isolation, purification, and applications, Appl. Biochem. Microbiol. 33 (1997) 111–115. [2] L. Lapˇcik Jr., L. Lapˇcik, S. De Smedt, J. Demeester, P. Chabreˇcek, Hyaluronan: preparation, structure, properties, and applications, Chem. Rev. 8 (1998) 2663–2684. [3] B. Fong Chong, L.M. Blank, R. Mclaughlin, L.K. Nielsen, Microbial hyaluronic acid production, Appl. Microbiol. Biotechnol. 66 (2005) 341–351.
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