Cephalosporin C 생산에 관한 동역학적 모사 및 혼합 종균 배양에의 응용

이명선, 김창호*, 오경근**, 김승욱

고려대학교 화학공학과, 버밍엄대학교 화학공학부 생물화학공학연구센타*,

단국대학교 공업화학과**

Modeling of Cephalosporin C production and

its application to the mixed seed culture

Myung Sun Lee, Chang Ho Kim*, Kyung Keun Oh**, Seung Wook Kim

Department of Chemical Engineering, Korea University,

Centre for Biochemical Engineering, School of Chemical

Engineering, The University of Birmingham*,

Department of Industrial Chemistry, Dankook University**

Introduction

Cephalosporin C (CPC) is a starting point in the manufacture of more potent cephalosporins for use in treating human infections caused by penicillinase-producing bacteria.

A kinetic model has been described for the production of CPC by considering morphological differentiation, induction of CPC production by methionine, and repression by glucose (Matsumura et al., 1981). Another mathematical model for batch fermentations has been developed using prototopic C. acremonium strain ATCC 3622S grown in a complex medium containing 25 g/L glucose and 30 g/L sucrose (Chu and Constantinides, 1988). Their model was used to determine optimal pH and temperature profiles from batch fermentation data.

In this study, the effect of seed mixture inocula on the production of CPC was examined by analyzing the morphological factors. Further, a kinetic model to describe the production of CPC was developed by considering morphological differentiation affected by inocula age.

Materials and methods

Organism and medium

Cephalosporium acremonium M25 was used in this study. Seed and main cultures were carried out at 27 ℃ on a rotary shaker at 300 rpm. Seed medium contained 2.5% sucrose, 1.0% glucose, 3.0% soy bean meal, 1.0% cotton seed flour, 2.5% corn steep liquor, 0.4% (NH4)2SO4, and 0.5% CaCO3, and  main medium contained 3.6% sucrose, 2.7% glucose, 0.8% (NH4)2SO4, 0.3% KH2PO4, 0.5% K2HPO4, 0.5% DL-methionine, 5% corn steep liquor and 0.4% trace element solution.

Analysis

Dry cell weight was used for cell mass, and modified  DNS method was used for measuring of residual glucose and sucrose concentrations. CPC was analyzed using the agar-dffusion paper disk method with Alcaligenes faecalis for bio-assay. Morphological differentiation was investigated with optical microscopy using Image Pro 3.0 software. Simulation was performed by a Runge Kutta method to solve ordinary differential equations using MATLAB 5.1 software.

Results and discussion

Structure of a kinetic model

The proposed model is based on the following experimental findings. Total cell mass was consisted of hyphae and swollen hyphal fragments ([eqn: X sub h] ), and arthrospores ([eqn: X sub a]). After depletion of glucose in the medium, swollen hyphal fragments shortened and finally differentiated into arthrospores. CPC was produced when swollen hyphal fragments differentiated into arthrospores. Glucose was used only as a energy source in growth phase, whereas sucrose was utilized only during CPC production phase.

By assuming that cellular differentiation is sequential and irreversible, the material balance of each type of cell could be expressed by the following equations.

[eqn: CHI = CHI sub h + CHI sub a]

[eqn: {d X sub h} over {dt} = {{mu} sub {hm} S sub G} over {K sub G + S sub G} X sub h - k sub dh X sub h - {{mu} sub {am} S sub G} over {K sub G + S sub G} X sub h]

[eqn: {dX sub a} over {dt}= {{mu} sub {am} S sub G} over {K sub G + S sub G} X sub h - k sub da X sub a]

where [eqn: X sub h] is the total cell mass of hyphae and swollen hyphal fragments, [eqn: {mu} sub {hm}] is the maximum specific hyphal growth rate, [eqn: K sub G] is the glucose saturation constant, [eqn: S sub G] is the glucose concentration, [eqn: k sub dh] is the specific hyphal death rate, [eqn: {mu} sub {am}] is the maximum specific arthrospore growth rate,[eqn: X sub a] is the total cell mass of arthrospores and [eqn: k sub da] is the specific arthrospores death rate.

[eqn: {dS sub G } over {dt}=- {1} over {Y sub X/G} ({{mu} sub {hm} S sub G} over {K sub G + S sub G} X sub h - k sub dh X sub h - {{mu} sub {am} S sub G} over {K sub G + S sub G} X sub h )- m sub s ( X sub h + X sub a )]

[eqn: {d S sub s} over {dt}=- {1} over {Y sub P/S } ( E X sub a -k sub pd P)]

where [eqn: {Y sub X/G}] is the cell mass yield factor from glucose, [eqn: m sub s] is the maintenance constant,[eqn: {Y sub P/S}] is the CPC yield factor from sucrose, [eqn: k sub pd] is the rate of constant of CPC decay and E is the amount of CPC-synthesizing enzymes.

Regarding to the formation of CPC-synthesizing enzymes, it assumed that the enzymes is formed by [eqn: X sub a] . Production of CPC was assumed to be proportional to the amount of enzymes.

[eqn: {dE} over {dt}= alpha {d{X sub a}} over {dt} - beta E]

[eqn: {dP} over {dt}= E X sub a -k sub pd P]

where □ is the growth-link enzyme formation rate, □ is the decomposition rate of the growth-link enzyme.

Parameter Evaluation

Parameters were estimated by two steps. At first, all parameters involved in growth, substrate consumption, enzymes and CPC production were introduced from the value of Matzumura et. al. (1981). However, the simulated values were not fit with the experimental values. From this results, it was thought that the estimated values ([eqn: k sub dh], [eqn: k sub da] and [eqn: k sub pd]) should be changed on the basis of the experimental results.

Simulation of mixed seed culture

The estimated parameter set was used to simulate the culture by using early-stage seed only as shown in Figure 1. Total cell mass and cell mass of hyphae were well fit between experimental and simulated results. But the cell mass of arthrospores was not properly simulated. It might be due to relatively lower value of the specific growth rate, comparing with the value obtained by Matzumura et al. (1981).

Figure 2 shows the experimental and simulated results of morphological differentiation using the late-stage seed only. Cell mass of hyphae and arthrospores were well fit between experimental and simulated results.

To predict the effective mixing ratio (v/v) of early and late-stage seed before experiments, CPC production was simulated according to various mixing ratio.

Figure 3 shows the simulated results of CPC production when the mixed seed of early and late-stage seed in various volume ratio were used. When 3:7 ratio of early and late-stage seed mixture was used as a inoculum, the maximum concentration of CPC produced was 3.08 g/L. Although the highest value of CPC concentration (3.10 g/L) was obtained at 1:9, the progress of CPC production was slower than the others. Also, the maximum productivity of CPC was obtained when 3:7 ratio of early and late-stage seed mixture was used.

Overall, simulation disclosed that differentiation of hyphae into arthrospores played a key role in CPC production. Also, simulation of mixed seed culture was successfully estimated.

Figure 1. Experimental and simulated    Figure 2. Experimental and simulated

results of morphological differentiation  results of morphological differentiation

using the early-stage seed only         using the late-stage seed only

Figure 3. Simulated CPC production with the mixed seed

of early and late-stage seed in various volume ratio

References

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3. Matsumura, M., Imanaka, T., Yoshida, T., and Taguchi, H. : J. Ferment. Technol., 59, 115-123 (1981)

4. Packer, H. L. and Thomas, C. R. : Biotechnol. Bioeng., 35, 870-881(1990)