Optimization of Dissolved Oxygen Supply Method for Maximum Virginiamycin Production

Copyright © IFAC Computer Applications in Biotechnology, Osaka, Japan, 1998

OPTIMIZATION OF DISSOLVED OXYGEN SUPPLY METHOD FOR MAXIMUM VIRGINIAMYCIN PRODUCTION Masatoshi Morikawa, Yoshinori Kajihara, Hiroshi Shimizu and Suteaki Shioya *

Department of Biotechnology, Graduate School of Engineering, Osaka University, Suita Osaka 565, JAPAN *Corresponding author. FAX:+81-6-879-7444 E-mail:[email protected]

Abstract. To maximize the production of virginiamycin (VM and VS) which is commercially used as an antibiotic mixed in animal feed, an empirical approach was employed in the batch culture of Streptomyces virginiae. Here, effects of Dissolved Oxygen (DO) concentration and/or DO supplying condition such as pure oxygen supply and agitation speed changes on the maximum cell concentration at the production phase as well as production activity of virginiamycin were investigated. In order to keep DO concentration in the fermenter at a certain level, either agitation speed or inlet oxygen concentration of supplying gas can be manipulated. It was found that the increase of agitation speed gave positive effect on antibiotics productivity apart from the controlled DO concentration. Optimum DO concentration and agitation speed including VB-C addition was determined for maximize virginiamycin production. Copyright © 1998 IFAC Key words: Streptomyces virginiae, autoregulator, virginiamycin fermentation, optimization

The dissolved oxygen (DO) concentration is also an important controlling parameter to be empirically decided. Oxygen limitation in accord with the increase of the cell concentration in the medium is well known to have a detrimental effect on cell activity and to decrease the productivity of antibiotics(Vardar and Lilly, 1982). Enhancement of antibiotics production by increasing the DO level has been investigated in penicillin and cephalosporin C fermentations, as well as others. Yegneswaran and Gray(Yegneawaran and Gray, 1991) reported a 2.4-fold increase in cephalosporin C production when the DO was controlled at a high level.

1. INTRODUCTION

Many studies regarding the optimization of antibiotics production in batch and fed-batch cultures have been reported(Bajapai and Reuss, 1981; Lim, et al., 1986; Mou and Cooney, 1983), most of which take a mathematical, model-oriented approach. However, empirical studies are also important for industrial process development because building a rigorous and reliable mathematical model requires much time or is almost impossible, and several empirically based studies of the growth phase have been published.

193

2.3 DO Control

In this paper, an empirical approach is also employed to maximize virginiamycin (M and S) production in a batch culture of Streptomyces virginiae. Here, effect of DO concentration and/or DO supplying condition such as pure oxygen supply and agitation speed changes on the maximum cell concentration at the production phase as well as production activity of virginiamycin is investigated and optimum DO concentration and agitation speed including VB-C addition was determined in order to maximize virginiamycin production.

A 5-Ljar fermentor (KMJ-5A, Mitsuwa Co., Japan) containing 1.5 L of medium was usually operated at 28°C, 450 rpm, and at a pH of 6.4-6.6. The dissolved oxygen (DO) concentration control was performed by two methods. One (called Method 1) is to adjust DO concentration mainly by manipulating the agitation speed but additionally by on-off flow control of pure oxygen in addition to air flow to the fermentor when the DO adjust by agitation could not give enough DO concentration . As shown in Fig.I, DO concentration decreases in accordance with cell growth as fermentation proceeds. When DO concentration reaches to setvalue, the agitation speed start to be manipulated to control the DO concentration. When the agitation speed reaches to the maximum value, 1000 rpm, and can not control DO concentration at a set-value, on-off flow control of pure oxygen is additionally employed. On the contrary, the other method (Method 2) mainly uses pure oxygen supply, that is, by on-off flow control of pure oxygen in addition to air flow to the fermentor but additionally by changing agitation speed when maximum addition of pure oxygen could not reach the desired DO concentration. In both cases, total inlet gas (air + pure oxygen) flow was kept at the constant 1 Umin. And minimum and maximum agitation speed were 450 rpm and l000rpm respectively. The following, Method 1 was usually used if not specify the method.

2. MATERIALS AND METHODS

2.1 Strain and Media Compositions Streptomyces virginiae MAFF 10-06014 (National Food Research Institute, Ministry of Agriculture, Forestry and Fisheries, Tsukuba, Japan) was used as Mand S-producing the vIrgJDlamycin microorganism. The culture medium used for virginiamycin production in this study consisted of 7.5 g/L Bacto-casitone (Difco Laboratories, Detroit, Ml), 45 g/L yeast extract (Difco), 15 g/L glycerol and 2.5 g/L sodium chloride. The pH was adjusted to 6.5. Components ofthe medium used to maintain the microorganism and the basic procedures for seed and batch cultures were as described previously(Yang, et al., 1995; Yang et al., 1996).

2.2 Analysis The cell concentration was measured as dry cell mass. The glycerol concentration was measured enzymatically and the virginiamycin M and S concentrations were determined by HPLC. The autoregulator concentration was measured as the VB-C concentration by an improved bioassay method(Yang, et al., 1995; Yang et al., 1996). The carbon dioxide concentration in the exhaust gas was monitored · as an indicator of the best timing for VB-C addition. These analytical procedures are described in detail in our previous paper(Yang, et al., 1995; Yang et al., 1996) .

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3. RESULTS AND DISCUSSION

3.1 Effect of DO concentration on the Productivity

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The DO concentration could be controlled at 50% of the saturation in a batch virginiamycin fermentation as shown in Fig.2 by DO control Method 1. DO control started after reaching 50%, that is, in the beginning of the batch culture DO was not controlled at 50% but higher than 50%. As is the characteristic feature of virginiamycin fermentation, around 1 to 2 hours before the start of virginiamycin production(lO hour), autoregulator measured as VB-C started to be produced. And the virginiamycin production phase, (after 11 to 12 hour), the specific growth rate was much smaller than the cell growth phase from the start of batch culture till 12 hours. As reported previously, S. virginiae produces virginiamycin M and S, which can act synergistically on microorganisms as antibiotics, but the ratio of virginiamycin M to virginiamycin S was found to be almost the same in all experiments done in this study. Moreover, because the amount of virginiamycin S produced is only about 10% that of virginiamycin M, in the subsequent experiments, attention is chiefly given to virginiamycin M production.

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Figure 3 shows the effect of DO concentration on the cell growth especially during the production phase. As can be seen in the figure, optimal level of DO concentration exist for getting maximum cell concentration. Here, DO concentration was controlled by Method 1. Agitation speed control started earlier as the control level of DO was higher. For 80% DO control, agitation speed reached at the maximum value. And additional control by pure oxygen supply was performed. Under too low level of DO concentration, it limits the cell growth whereas under high level of DO concentration, rather higher agitation may cause the decrease of cell growth rate. However, for maximizing productivity of virginiamycin, higher level of DO concentration is desired as shown in Table 1. Maintaining at 80% level of DO concentration gave about 300 mgIL virginiamycin-M(VM) production which is about 1.4 times higher than the maximum value previously obtained. (Yang, et al., 1995; Yang et al., 1996)

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Table 1

3.2 Positive Effect of Agitation speed on the Productivity In those experiments(fable 1), DO concentration was controlled mainly by the agitation speed. It means that a high DO concentration corresponds to a high agitation speeds. Then, the question which event, high DO concentration or high agitation speed is responsible to a high virginiamycin productivity will arise. Now, the following experiment had been performed. That is, the profile of agitation speed change had been controlled to be almost the same as those in 50% DO concentration but the controlled DO concentration was lower than 50%, which was really 25%. Such a experiment could be performed by mixing N2 gas in addition to air flow to the fermentor.

Productivity of virginiamycin at various control level of DO Concentration 10%

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It was found that both agitation speed and DO concentration in the fermentor have significant positive effect for productivity of virginiamycin and autoregulator. Positive effect of DO concentration on productivity may not be new finding but the positive effect of the agitation speed on the productivity has not been proved in such a way shown here and will be a new evidence. The same evidence can be seen at high DO concentration and high agitation speed. From the comparison of these data, again it can be found that both agitation speed and DO concentration enhance the virginiamycin productivity.

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It was found that the agitation speed enhance the production activity. This is apart from due to DO concentration increase but agitation itself. The true reasoning is not clear now but our speculation is somewhat due to micromixing around cell related to the autoregulator distribution. AutoreguI ator is understood now as a part of signal peptide for signal transduction to start of virginiamycin production. Autoregulator will have complex function such as too much addition inhibit the virginiamycin production. The reasoning for positive effect of agitation will be left further study.

It was shown that not only the DO concentration but also the agitation speed itself enhance the virginiamycin production. However, as already mentioned, much higher agitation speed decreases the cell growth rate. The cell concentration at the production phase of virginiamycin is related to the productivity of VM and vs. Finally, the optimal agitation speed might exist. Figure 5 shows the effect of upper limit of agitation speed on the virginiamycin productivity. From the figure, an optimal upper limit of agitation speed is found to be 800 rpm. Of course to keep rather high agitation speed such as 800 rpm from the start of batch culture was not good for cell growth as well as virginiamycin production. So the optimal strategy is: to start at 450 rpm and continue till DO concentration became less than 80%. After reaching at 80 %, to control DO concentration at 80% by Method 1 where the maximum agitation speed is set at 800 rpm.

In addition to control the DO concentration by Method 1, to enhance more the production activity, addition of an appropriate amount of the autoregulator VB-C at an appropriate time was examined. In the previous study, the important evidence was: there exists the optimum autoregulator concentration per cell (VBs mglg cell). By addition of VB-C, the maximum production of virginiamycin M was about 400 mgIL which is about 1.8-fold that obtained previously.

REFERENCES

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Bajpai, R.K.and M. Reuss (1981). Evaluation of feeding strategies in carbon-regulated secondary metabolite production through mathematical modelling. Biotechnol. Bioeng. 23, 717-738. Lirn, H.C. ,YJ. Tayeb, I.M. Modak and P. Bonte. (1986). Computational algorithms for optimal feed rate for a class of fed-batch fermentation: Numerical results for penicillin and cell mass production. Biotechnol. Bioeng. 28: 1408-1420 Mou, D.G.and C.L. Cooney. (1983). Growth monitoriJlg and control in complex medium: A case study employing fed-batch penicillin fermentation and computer-aided on-line mass balancing. Biotechnol. Bioeng. 25, 257-269 Vardar, F. and M.D. LiIly. (1982). Effect of cycling dissolved oxygen concentrations of production formation in penicillin fermentation . Eur. 1. Apply. Microbiol. Biotechnol. 14,302-311

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Yegneswaran, P.K. and M.R. Gray. (1991). Effect of dissolved oxygen control on growth and antibiotic production in Streptomyces clavuligerus fermentation. BiotecMol. Prog. 7,246-250 Yang, Y.K., H. Shimizu, S. Shioya, K. Suga, T. Nihira and Y. Yamada. (1995). Optimum autoregulator addition strategy for maximum virginiamycin production in batch cultivation of Streptomyces virginiae. Biotechnol. Bioeng. 46, 437-442 Yang, Y.K., M. Morikawa, H. Shimizu, S. Shioya, K. Suga, T. Nihira and Y. Yamada. (1996). Maximum virginiamycin production by optimization of cultivation condition in batch culture with autoregulator addition. BiotecMol. Bioeng. 49,437-444

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