플라스크 진탕배양기의 운동방향이 미생물 셀룰로오스의 생산에 미치는 영향

김대훈, 이희찬

선문대학교 화학공학부 생물공정실험실

The Effect of Mode of Shaking Flask Incubator Motion on the Production of Bacterial Cellulose: Rotary Shaking and Reciprocal Shaking

Dae Hoon Kim, Hei Chan Lee

Bioprocess Engineering Laboratory, Division of Chemical Engineering

College of Engineering, Sun Moon University

Introduction

Bacterial Cellulose (BC, microbial cellulose: MC) appears as a form of ribbon-like fibrils in contrast to the cellulose of high plants of consisting of bundles of microfibrils. It possesses excellent physical and chemical properties, such as strong mechanical properties, high degree of polymerization and is composed of chemically pure cellulose without lignin, hemicellulose, and other substances[1-3]. At present, the utilization of these properties has been studied for several aspects of practical usage of bacterial cellulose[4], for examples, sensitive diaphragms for stereo headphones, additives for food and paper products, thickener for paints, and also artificial skin in skin burn treatment[5-10,11].

However, industrial development of these production process has been hampered by the fact that genetic variations with reduced or no production of bacterial cellulose can be caused by shaking or agitation. Therefore, investigations not only the optimum composition of the medium[12,14,15], but also environmental conditions for the production under shaking culture conditions[13,16,17], such as optimum oxygen transfer rate, effect of directional shear, micro-environment and agitation speed, and so on. In this paper, the effect of the mode of shaking incubator motion on the yield of bacterial cellulose was studied by different type of shaking incubators.

Materials and Methods

In this experiment, Acetobacter xylinum BRC 5, provided by Bioproducts Research Center of Yonsei University, was used for the production of bacterial cellulose. The concentrated cells was preserved at -80。C with glycerol. After innoculation into Hestrin-Schramm medium[13] composed of 10g peptone, 20g yeast extract, 5g glucose, 1.56g Na2HPO4, 1.8g KH2PO4, 0.05g MgSO4, 0.002g FeCl3, 5g citric acid and 10ml ethanol per liter, it was cultured at 30。C for 6 days in a conical tube without shaking. Then, cellulose pellicle formed on the air-liquid interface was homogenized for 2 minutes by homogenizer(ESGE, M133/1280-0), and the homogenate was used as an inoculum for the flask culture.

In this study, two types of shaker was used to investigate the variation of the yield according to the shaking type. One is rotary shaking type, Rotary Shaking Incubator (Vision, KMC-8480SF), and the other is reciprocal shaking type, Shaking Water Bath (Vision, KMC-1205SW1). For the two type of incubators, the yield of bacterial cellulose was measured at rotation speeds, 50 rpm, 150 rpm, 200 rpm, and 50 rpm, 100 rpm, 150 rpm, respectively.

Six days after the incubation, the culture was collected and the product was filtered over paper filter(ADVANTEC, 110mm). Then it was stored in the distilled water for soaking and transferred to a fresh distilled water two more times, and it was boiled in 4% NaOH solution for 15 minutes to remove the cells components in cellulose pellicle. And then it was soaked again with distilled water to remove the trace of NaOH. The cleaned cellulose product was dried at 80°C overnight, then cooled to room temperature, and the weight of the dried cellulose was measured.

Result and Discussion

The effect of shaking type on the yield of cellulose was investigated under shaking speed of 50rpm, 150rpm and 250rpm for rotary shaker, 50rpm, 100rpm and 150rpm for reciprocal shaker. Volume fractions applied were 0.1, 0.2, 0.3 and 0.4 and volume fraction was defined as culture volume divided by the capacity of flask.

Figure 1 shows the difference in bacterial cellulose yield at the rotation speed of 50 rpm between two types of shaker. The yield of cellulose for rotary shaker was superior to the reciprocal shaker at all volume fractions from 0.1 to 0.4. In rotary shaker, the yield decreases as volume fraction increases. However, in reciprocal shaker, the yield slightly increases as volume fraction goes to 0.2 then decreases at 0.3 and 0.4. At 50 rpm of rotation and reciprocal shaking motion, oxygen transfer rate difference is not large enough to affect the production of bacterial cellulose since the surface did not form vortex at that row shaking speed. Figure 2 shows that the yields of rotary shaker are generally higher than those of reciprocal shaker. In rotary shaker, as shaking speed increase the yield generally increases up 250 rpm and decreases as volume fraction increases. In reciprocal shaker, the cellulose yield of 100 rpm is higher than that of 50 rpm and the yield of 150 rpm is lower than that of 100 rpm but still higher than that of 50 rpm. This observation coincide with other report[16] and the yield peak at 100 rpm in reciprocal shaker explains more clearly about the effects of directional shear stress. At 150 rpm in reciprocal shaker, shaking motion provides non-directional shear stress to the cells producing bacterial cellulose which may be caused genetic variations. The relationship between the yield of bacterial cellulose and cel- mutant which does not produce bacterial cellulose was reported earlier[17]. The effect of increasing shaking speed on the production of bacterial cellulose was found to be affirmative up to a certain point. This explains that oxygen transfer is an important factor for the production of bacterial cellulose by Acetobacter xylinum which floats to the surface under static culture condition. That is how they get the access th the oxygen under the static culture condition and, as many researchers suggest, why they produce cellulose as a form of pellicle attached to themselves. At 150 rpm in reciprocal shaker, the liquid environment seems to generate shear stress high enough to invoke genetic variations to the cells.

Through this study, we could obtain the informations about the culture conditions of Acetobacter xylinum for the production of bacterial cellulose, such as agitation type or agitation speed, for the larger scale of BC fermenter, which should be interpreted as proper numbers of fluid mechanical terms.

Figure1. The yield of cellulose at 50rpm

Figure 2. Comparison of yield between rotary and reciprocal type shaker

Acknowledgement

This study is supported partly by the academic research fund of Ministry of Education , Republic of Korea.

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