THE EFFECT OF THE ADDITION OF INVERT SUGAR ON THE PRODUCTION OF CEPHALOSPORIN C IN A FED-BATCH BIOREACTOR

THE EFFECT OF THE ADDITION OF INVERT SUGAR ON THE PRODUCTION OF CEPHALOSPORIN C IN A FED-BATCH BIOREACTOR

A.S. SILVA², A.J.G. CRUZ², M.L.G.C. ARAUJO1,^* and C.O. HOKKA²

¹Universidade Estadual Paulista, Instituto de Química, Departamento de Tecnologia, P.O. Box 355, 14801-970, Araraquara - SP, Brazil - Phone (PABX) [phone], FAX [phone], E-mail: [e-mail]

^‡ Universidade Federal de São Carlos, Departamento de Engenharia Química, P.O. Box 676, 13565-905, São Carlos - SP, Brazil - Phone [phone], FAX [phone]

(Received: May 10, 1998; Accepted: August 24, 1998)

Abstract - Cephalosporin C, a b-lactam antibiotic, is the starting molecule for industrial production of semi-synthetic cephalosporins. The bioprocess for its production is carried out in batch stirred and aerated tank reactors utilizing strains of the filamentous fungus Cephalosporium acremonium. In this work a comparison was made between the processes of production of cephalosporin C in a conventional batch bioreactor, with synthetic medium containing glucose and sucrose, and in a fed-batch reactor at several flowrates of supplementary medium containing invert sucrose. In general, the fed-batch process was shown to be more efficient than the conventional batch one, and the process in which the lowest supplementation flowrate was used presented an antibiotic production significantly higher than those obtained under the other conditions.

Keywords:Cephalosporium acremonium, invert sugar, cephalosporin C, fed batch.

INTRODUCTION

Cephalosporin C is a b-lactam antibiotic which, like all secondary metabolites, is synthesized in bioprocesses involving microorganisms. Industrial production of this antibiotic is still carried out in conventional batch fermentations in aerated stirred tank bioreactors by means of submerged cultures of mutant strains of Cephalosporium acremonium.

Regulatory mechanisms for secondary metabolism in this process include repression and inhibition of the b-lactam synthetases by glucose and other carbon and energy sources easily metabolized by the microorganism. These compounds are essential for cell growth, but drastically suppress the production of many antibiotics (Zanca and Martin, 1983; Vicik et al., 1990).

In the batch process of the production of cephalosporin C by C. acremonium in a synthetic medium containing glucose and sucrose as the carbon and energy sources, the diauxic phenomenon is observed. It is characterized by the rapid consumption of glucose to form biomass (trophophase) at the beginning of the process and, after depletion of this carbohydrate, by the slow consumption of sucrose, the more difficult carbohydrate to assimilate, during which the majority of the cephalosporin is produced (idiophase) and cell growth is insignificant. The slow metabolism of sucrose may be explained by the low activity of the enzyme responsible for the hydrolysis of this sugar during the process (Vicik et al., 1990; Machado et al., 1997).

One of the main advantages of the fed-batch process in relation to the simple batch process is the possibility of increasing antibiotic production by addition of substrates at a suitable flowrate. Several types of carbohydrates even those which are easily assimilated, may be used as an adequate feed strategy allows the effects of catabolite repression on the b-lactam synthetases to be diminished. However, even when added slowly, sucrose tends to accumulate in the reaction medium in virtue of the low activity of the enzyme that hydrolyzes this carbohydrate, thereby limiting its consumption rate (Gomes et al., 1996; Cruz et al., 1998).

The use of sucrose is, therefore, a good strategy to obtain high antibiotic concentrations in the conventional batch process, but is unsuitable for the fed-batch process. A viable alternative is to use hydrolyzed sucrose in the supplementary medium which, together with the low cost of this sugar on the Brazilian market in relation to other carbohydrates such as glucose and fructose, may make the fed-batch process even more advantageous.

In the current work an investigation was performed on the effect of feed rate on the production of cephalosporin C in an aerated stirred tank reactor, using hydrolyzed sucrose in the supplementary medium. The establishment of flowrates was based on the sucrose consumption rate observed in the conventional batch process. Addition of the substrate at suitable flowrates avoided accumulation of carbohydrates in the reaction medium, as well as catabolite repression in the synthesis of the antibiotic.

MATERIALS AND METHODS

Microorganism

Cephalosporium acremonium ATCC 48272 (C-10), kindly donated by the Fundação Tropical André Tosello (Campinas-SP) and stored in solid medium (Shen et al., 1986), was used.

Synthetic Culture Medium

Germination and inocula preparation (Demain et al., 1963; Araujo, 1996) (in g/l):

Glucose (30.0), oleic acid (1.5), DL-methionine (3.0), ammonium acetate (8.8), KH₂PO₄ (2.3), K₂HPO₄ (5.8), Fe(NH₄)₂(SO₄)₂.6H₂O (0.16), CaCO₃ (2.0), 50 ml concentrated salt solution containing (g/l) Na₂SO₄ (16.2), MgSO₄.7H₂O (7.68), CaCl₂.2H₂O (7.68), MnSO₄.H₂O (0.64), ZnSO₄.7H₂O (0.64), CuSO₄.5H₂O (0.004); pH = 7.0 ± 0.1.

Main fermentation (Araujo, 1996):

The medium for the main fermentation of the simple batch process contained (in g/l) glucose (27.0), sucrose (36.0), KH₂PO₄ (1.8), K₂HPO₄ (2.97), and the remaining components in the same amounts as those in the precultures (germination and inoculum preparation).

The composition of the initial batch medium of the semi-continuous process was the same as that used in the conventional batch process (Run #1), excluding sucrose. The supplementary medium utilized in Runs #2, #3 and #4 contained the same components as the initial batch and previously hydrolyzed sucrose and resulted in carbon source concentration (in g/l) of glucose (77.5) and fructose (50.5) in Run #2, glucose (69.6) and fructose (42.6) in Run #3 and glucose (89.7) and fructose (62.7) in Run #4.

The sucrose used in the supplementary medium was hydrolyzed at 40° C in a buffer solution of 10^-2M sodium acetate at a pH of 4.5, employing invertase enzyme in soluble powder form, which was kindly supplied by Novo Ferment (Ribeiro, 1989).

Analytical Methods

Cell concentration:

Expressed as volatile solids in suspension (VSS).

Glucose and sucrose concentration:

Enzyme-colorimetric GOD-PAP (glucose oxidase) method; for sucrose determination, samples had been previously hydrolyzed in acid medium.

Cephalosporin C concentration (CPC):

Obtained by agar diffusion bioassay, using Alcaligenes faecalis strain ATCC 8750 as the test bacterium.

Experimental Procedure

The fermentation runs were carried out in an aerated 5.0 l stirred tank bioreactor (Bioflo III, New Brunswick) equipped with instruments for temperature and dissolved oxygen control and monitor, and a pH meter. The reactor exit gas was attached to O₂ paramagnetic (Rosemount Analytical Model 755) and CO₂ infrared (Rosemount Analytical Model 880A) analyzers.

All runs were carried out at 26° C for periods ranging from 137 to 144 hours. The inoculum of the main fermentation medium was standardized for all experiments at 10% vol. inoculum/vol. initial fed-batch medium (ca. 300 ml inoculum). The dissolved oxygen concentration was maintained at a 27% saturation by automatic variation of the stirring speed and the air flowrate used (ca. 3 l air/min).

The conventional batch fermentation (Run #1) contained 4.5 l of main fermentation medium. The fed-batch fermentations, Runs #2, #3 and #4, were started with 3 l of medium and, after depletion of the glucose, supplementary medium was added at established flowrates until 4.5, 5.0 and 4.0 liters, respectively, were attained at the end of 100 hours of feed supply.

The purpose of the first fed-batch experiment, Run #2, was to reproduce the sucrose consumption rate during the idiophase of the conventional 4.5 l batch run (Run #1). The hydrolyzed sucrose feed rates of the remaining runs were established close to the value used in Run #2, which was adopted as the reference run. The general operational conditions are listed in Table 1.

Table 1:
Operational conditions of the fed-batch runs

Run	#2	#3	#4
Final volume (l)	4.5	5.0	4.0
Flowrate (ml/h)	15.4	20.0	10.3

The instant of feed starting was estimated by determining the glucose concentration in the fermentation medium. Samples of ca. 15 ml were periodically withdrawn and pH, cell concentration, sugars and cephalosporin C were analyzed.

RESULTS AND DISCUSSION

Figure 1 illustrates the conventional batch experimental data (Run #1). Diauxic behaviour is evidenced by the preferential consumption of glucose for the formation of biomass and subsequent assimilation of sucrose during the production stage. Cell growth took place at approximately 47 hours, resulting in a maximum value of about 15.0 gcel/l. Synthesis of cephalosporin C increased up to approximately 120 hours of processing time, attaining ca. 1.2 gCPC/l, and then decreasing together with the cell concentration, in virtue of substrate depletion.

All fed-batch runs are illustrated in Figures 2 to 4. Analysis of the results for cell concentration shows that the maximum values are reproducible and are close to those for the biomass in the conventional process, about 15 gcel/l. Due to the addition of the medium, cell concentration of the fed-batch fermentation remained practically constant and near its maximum value during the entire production phase. To better illustrate this, these results were plotted together in Figure 5.

Figure 1: Experimental results of glucose, sucrose, biomass and cephalosporin C (CPC) concentrations in the conventional batch fermentation (Run #1).

Figure 2: Concentration profiles of glucose, biomass and cephalosporin C (CPC) during Run #2,

fed-batch fermentation with feed flowrate of 15.4 ml/h.

Figure 3: Concentration profiles of glucose, biomass and cephalosporin C (CPC) during Run #3,

fed-batch fermentation with feed flowrate of 20.0 ml/h.

Figure 4: Concentration profiles of glucose, biomass and cephalosporin C (CPC) during Run #4,

fed-batch fermentation with feed flowrate of 10.3 ml/h.

Figure 5: Biomass concentration profiles during the conventional batch and fed-batch runs.

Figure 6: Comparison of the production of cephalosporin C (CPC) during the four runs performed.

Depletion of glucose and the subsequent start of the idiophase in the fed-batch runs were anticipated in relation to the simple batch fermentation. Addition of supplementary medium was started after about 37 hours fermentation at all flowrates investigated. Analyses of glucose and fructose concentrations in the fermentation medium during the feeding stage of these sugars indicated values close to zero, with no significant differences under the three conditions investigated.

The concentration profiles of cephalosporin C were plotted together in Figure 6. The fed-batch production rates are somewhat higher than those of the conventional batch process.

In Run #2, with the operational conditions equivalent to the conventional batch of 4.5 liters (Run #1), the maximum cephalosporin C concentration was on the order of that obtained in the conventional batch fermentation, i.e., ca. 1.1 gCPC/l, the difference being that this concentration was attained after 75 hours of fermentation and remained practically constant during the entire feeding stage. The specific productivity, estimated at about 1.2 mgCPC/g cel.h, was about 2.3 times higher than that of the conventional batch process.

Run #3 was performed at a flowrate 30% higher than that in Run #2 and the average cephalosporin C concentration obtained, 0.84 gCPC/l, was approximately 30% less. The results suggest the occurrence of significant catabolite repression due to the higher mass of sugars supplied in relation to the former condition.

The last feed condition investigated (Run #4) reproduced the production phase of the conventional batch process for 4.0 liters, supplying hydrolyzed sucrose at a flowrate about 48% lower than that in Run #2. In Figure 5 high production rates can be observed after 40 hours, with 1.5 gCPC/l obtained after 70 hours of fermentation. After this point production can be seen to be increasing, although at lower rates, attaining 1.9 gCPC/l at the end of the process without any signs of apparent degradation of the antibiotic.

The results obtained showed high production rates and upkeep of the maximum cell concentration during the entire idiophase under all feeding conditions. Of the several conditions studied resulted in a significantly higher productivity in the lowest flowrate (Run #4), certainly due to lower catabolite repression. Although it was not possible to detect remarkable differences in glucose concentration, inside the cells, a control through regulation by CAP (catabolite activator protein) type molecules probably occurred. As has been observed for lac-operon (Darnell et al., 1990), its activation property was controlled by different levels of cAMP depending on the glucose/fructose feed rate. A lower flowrate may still be investigated, taking care to maintain the integrity of the cell population by supplying a minimum of the remaining nutrients.

CONCLUSIONS

The increase in production of cephalosporin C achieved as the flowrate of supplementary medium is reduced in the fed-batch fermentation process and the influence it exerts on the biosynthesis of the antibiotic are evident in the results obtained.

In general, slow addition of supplementary medium containing hydrolyzed sucrose resulted in a reduction in catabolite repression and was shown to be a much more advantageous strategy than the conventional batch process with sucrose.

The fed-batch fermentation favoured maintenance of higher antibiotic production rates during the process in relation to those obtained in the batch process, resulting in a higher specific production.

ACKNOWLEDGMENTS

A.S.S. is grateful to CNPq. A.J.G.C., C.O.H. and M.L.G.C.A. acknowledge the financial support of FAPESP (Proc. 96/3170-7, 96/5918-9 and 96/10.010-6, respectively).

References

• Araujo, M.L.G.C., Estudo Cinético do Processo de Produçăo de Cefalosporina C com Células Livres e Imobilizadas de Cephalosporium acremonium ATCC 48272. Departamento de Engenharia Química, Universidade Federal de Săo Carlos, Săo Carlos, SP, (Ph.D. thesis), 189p. (1996).
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• Gomes, L.M.S.; Hokka, C.O. and Santana, M.H.A., Bioprocesso em Batelada Alimentada para Produçăo de Cefalosporina C. Anais 11^o Congresso Brasileiro de Engenharia Química (XI COBEQ), Rio de Janeiro, RJ, vol. 2, 1514-1519 (1996).
• Machado, J.R.; Araujo, M.L.G.C. and Hokka, C.O., Estudo Cinético da Hidrólise da Sacarose no Processo de Produçăo de Cefalosporina C. Livro de Resumos do XIX Congresso Brasileiro de Microbiologia, p. 200, Rio de Janeiro, RJ (1997).
• Ribeiro, E.J., Estudo Cinético da Hidrólise de Sacarose por Invertase Livre e Imobilizada. Faculdade de Engenharia de Alimentos, Universidade Estadual de Campinas, Campinas, SP, (Ph.D. thesis), 202p. (1989).
• Shen, Y.-Q.; Wolfe, S. and Demain, A.L., Levels of Isopenicillin N Synthetase and Deacetoxycephalosporin C Synthetase in Cephalosporium acremonium Producing High and Low Levels of Cephalosporin C. Bio/Technology, vol. 4, pp. 61-63 (1986).
• Vicik, S.M.; Fedor, A.J. and Swartz, R.W., Defining an Optimal Carbon Source/Methionine Feed Strategy for Growth and Cephalosporin C Formation by Cephalosporium acremonium Biotechnol. Prog., vol. 6, n^o 5, pp. 333-340 (1990).
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*

To whom correspondence should be addressed.

Publication Dates

• Publication in this collection
  07 Dec 1998
• Date of issue
  Dec 1998

History

• Accepted
  24 Aug 1998
• Received
  10 May 1998