Cephalosporin C 생산성 향상을 위한 Cephalosporium acremonium의

형태학적 연구

이명선, 김창호*, 김승욱

고려대학교 화학공학과, 버밍엄대학교 화학공학부 생물화학공학연구센타*

Morphology study of Cephalosporium acremonium for the

Improvement of Cephalosporin C Productivity

Myung Sun Lee, Chang Ho Kim*, Seung Wook Kim

Department of Chemical Engineering, Korea University,

Centre for Biochemical Engineering, School of Chemical

Engineering, The University of Birmingham*

Introduction

The morphology of filamentous microorganisms in submerged culture is of great interest because in many cases productivity correlates with morphology of filamentous microorganisms such as Streptomyces and fungi.

In submerged culture, Cephalosporium acremonium shows three types of morphological differentiations; hyphal filaments, swollen hyphal fragments, and arthrospores.  Production of Cephalosporin C(CPC) is too closely related with the morphological differentiation of C. acremonium.  In this study, the morphological factors such as total hyphal length, main hyphal length, number of tips and number of arthrospores were observed to identify the relationships between seed morphology and CPC production.

Materials and methods

Organism and medium

The Cephalosporium acremonium M25 was used in  this study. The stock cultures were maintained on PDA(potato dextrose agar) slants at 4℃ after 7-day incubation at 27 ℃. Seed and main cultures were carried out at 27 ℃ for 3 days 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. The composition of trace solution was shown in Table 1.

Table 1. The composition of trace element solution

Component	Concentration(g/L)
CaCl2-2H2O	0.07
CuSO4-5H2O	0.0075
ZnSO4-7H2O	0.03
MnSO4-H2O	0.03
MgSO4-7H2O	0.35
Na2SO4-10H2O	1.65
Fe(NH4)2(SO4)2-6H2O	0.16

Analysis

PMV(packed mycelia volume) 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 factors were investigated with optical microscopy using Image Pro 3.0 software.

Results and discussion

Typical morphological changes in seed cultures were shown in Figure 1. Early-stage seed broth of 2 days(Figure 1(a)) had many filamentous hyphae and a few swollen hyphal fragments, but no arthrospore. In  seed broth of 4 days(Figure 1(b)), some filamentous hyphae and lots of swollen hyphal fragments were observed. A few arthrospores were also produced. However, there were  many swollen hyphal fragments and arthrospores in the late-stage seed broth of 6 days(Figure 1(c)).

These results mean that the characteristics of morphological factors are quite different due to the inoculum age of seed culture.

(a)                             (b)                             (c)

Figure 1.  Morphology of Cephalosporium acremonium M25 in seed culture;

(a) 2 days (b) 4 days (c) 6 days

The process of image analysis using Image Pro Plus software was shown in Figure 2. Measurements of multiple objects within a single image were collected on numerical data with the tools on the Measure menu(Figure 2(a)). The objects were automatically counted after sorting and classifying by any of the measured characteristics(Figure 2(b)).

(a)                                           (b)

Figure 2. Image processing for (a) measurement of hyphal length, and

(b) counting of arthrospores

Growth curve of C. acremonium M25(Figure 3) shows the trend of consumption of sugars, cell mass and pH. Glucose was rapidly utilized and depleted during growth phase whereas sucrose was utilized only during CPC production phase(idiophase). Cell mass was maintained from 24 to 27%(PMV). PH was decreased up to 2 days, and then increased to 8.42. The decrease of pH might be due to the formation of intermediates such as citric acid.

The time course of morphological factors of C. acremonium M25 in the seed culture by image analysis was shown in Figure 4. Main hyphal length was shortened from 83□ to 4□ and the number of arthrospores increased to 2.95x104/mL for 7 days. In results, total hyphal length and main hyphal length were rapidly shortened and arthrospores was increased according to time. Therefore, we found that the age of inoculum could be resulted in serious effects during the main culture.

Using the analysis of the morphological factors in the seed culture, the effect of inoculum age of seed culture on the production of CPC was carried out in the main culture. The results were shown in  Figure 6.

CPC production of early-stage inoculum(seed of 2 days) relatively prolonged compared with intermediate-stage inoculum(seed of 4 days) and late-stage inoculum(seed of 6 days). In the case of early-stage inoculum, the level of CPC production was slower than the others till 2 days. However, the maximum production of CPC was 2.04g/L in 5 days, and this was the highest value compared with the cases of other stages. In the case of  late-stage inoculum, CPC production reached at the maximum value within 3 days, and then decreased gradually.

From these results, the case of early-stage inoculum was found to be a proper seed condition, considering production and stability of CPC.

Overall, we obtained the morphological data by image analysis and found that the morphology of C. acremonium played an important role in CPC production. Thus, morphology control of seed culture could be enhanced the productivity of CPC.

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