Cephalosporium acremonium M25 배양액의 형태학적 특성 및 유변학적 특성연구

임정수, 김진희, 홍석인, 김승욱

고려대학교 화학공학과

Morphological and rheological properties of culture broth of Cephalosporium acremonium M25

Jung Soo Lim, Jin Hee Kim, Suk In Hong and Seung Wook Kim

Department of Chemical Engineering, Korea University

1. Introduction

Cephalosporium acremonium, an imperfect fungi, is an object of constant interest on account of its potential use as a producer of Cephalosporin and enzymes. The morphological differentiation of C. acremonium in submerged culture is closely related with the production of Cephalosporin C. Also the rheological behavior of a fermentation broth is of considerable importance in describing the transport phenomena in a fermentor. In this study the rheological model applicable for the description of the flow behavior of the fermentation broth was examined and the effect of  mycelial morphology on the rheological behavior of the broth was also investigated.

2. Materials and methods

2.1 strain

The microorganism used in these study was mutant strain, C. acremonium M25, treated with UV.

2.2 Media and culture conditions

Basal seed medium consisted of 2.5% sucrose, 1.0% glucose, 2.5% corn steep liquor and 0.4% (NH4)2SO4. Seed cultures were carried out in 250 □ Erlenmeyer flasks containing 50 □ of medium. The shake-flask cultures were operated at 300 rpm, 27℃, on a rotary shaking incubator. The batch fermentation in the stirred-tank fermenter (2.5 L) was carried out at 27℃. Operating volume was 2.0 L and air flow rate was 1.0 vvm.

2.3. Analysis

2.3.1 Image analysis for cell morphology

The cell morphology was studied on photomicrographs with optical microscopy connected with Image Pro 3.0 software.

Morphological factors such as hyphal length, number of tips, number of arthrospores and length of swollen hyphal fragments were measured manually or automatically after sorting and classifying by image analyzing process.

2.3.2 Analysis of rheological parameters

The Brookfield LVT viscometer was used to determine the shear stresses(□) and apparent viscosities at different shear rates ().

3. Results and Discussion

3.1 Morphological changes of C. acremonium

Typical morphological changes during 6 days in the seed cultures were taken by a camera mounted on an optical microscope (Fig.1). Morphological characteristics between shake-flask and fermenter cultivation show different tendency in differentiation. In shake-flask cultivation, seed broth of 2 days contained many filamentous hyphae and a few swollen hyphal fragments, but no arthrospore. But, in fermenter cultivation, there were many swollen hyphal fragments and the progress of morphological differentiation was faster than in shake-flask cultivation. In both seed broth of 4 days, some filamentous hyphae and lots of swollen hyphal fragments were observed. Swollen hyphal fragments in fermenter cultivation were thicker than in shake-flask cultivation. There were also many swollen hyphal fragments and arthrospores in both seed broth of 6 days.

3.2 Rheological model applicable to the mycelial broth of C. acremonium

Figs. 2, 3 and 4 show the shear stress versus shear rate plots made by different rheological equations expressing the flow behavior of fermented broths. The power law is in excellent agreement with result of culture broth from shake-flask culture in the whole range of shear rates (Fig.2-a). But Fig.3-b shows that  the experimental results in the fermenter culture agree well with Bingham plastic equation. Herschel-Bulkley plot of shear stress against shear rate is shown in Fig. 4. It shows some deviations at the whole range of shear rate tested.

4. Conclusion

From the analysis of the morphological characteristics, seed broths from shake-flask and fermenter cultures show very different morphological pattern. It is therefore thought that different culture conditions could affect to productivity of the CPC. In analysis of rheological model applicable to the seed broth, it showed actually different flow pattern. Power law equation agreed with flow pattern of culture broth from shake-flask culture. But fermenter broth showed   the flow pattern of Bingham plastic.

5.Acknowledgement

This study was partially supported by research grants from the KOSEF through Applied Rheology Center at Korea University, Korea.

6. Reference

1. G. L. Riely and C. R. Thomas, "Effect of biomass concentration and mycelial morphology on fermentation broth rheology", Biotechnol. Bioeng., 68, 160-172 (2000).

2. A. Mitard and J. P. Riba, □"Rheological properties of Aspergillus niger pellet suspensions." Appl. Microbiol. Biotechnol., 25, 245-249 (1986).

Figure 1. Typical morphological changes in the flask cultivation and fermenter cultivation of C. acremonium M25.

Fig.2 Shear stress vs. shear rate plots made by the power law equation for the expression of the flow behavior of seed broth at different cultivation time.

Fig.3 Shear stress vs. shear rate plots made by the Bingham plastic equation for the expression of the flow behavior of seed broth at different cultivation time.

Fig.4 Shear stress vs. shear rate plots made by the Herschel-bulkley equation for the expression of the flow behavior of seed broth at different cultivation time.