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1 The Krean Jurnal f Micrbilgy, Vl. 45, N. 3, September 009, p Cpyright 009, The Micrbilgical Sciety f Krea e w Acetbacter sp. V6 y ½ Á y Á wá»xá Áy Á Á y * w w w w m z l Acetbacter sp. V6 w bacterial cellulse (BC) w e w w z, BC p mw. k glucse 3%, sytne 3%ƒ. w K H 0.8%, ƒ ƒw NaH KH BC ƒ ùkü w. k ethanl 0.4% ƒ BC ùkü. w g/m BCƒ, 8, BC pellicle Ì 1cm. BC Furier-transfrm infrared spectrscpy X-ray diffractmeter w w. BC x cellulse type I, ƒ. w pw š, ùkü pw ùkü. Key wrdsý Acetbacter sp., bacterial cellulse, cellulse type I, static culture ƒ t w ƒ w glucseƒ β-1, 4 w w š. x, rv w w š ƒ j ƒ š. yw t e w w w, š, w l, p l y š (1, 10, 0). x w š ù œ y w w ƒ y w š (4, 1). Agrbacterium, Pseudmnas, Rhizbium Acetbacter ³ y, Acetbacter spp. ƒ w š (1). w ³ (bacterial cellulse; BC) pw ƒ š š, w» y w» w ƒ y w š (, 9, 6). BC hemicellulse, pectin, lignin š bigenic prduct x sww w t š w (15, 5). š BC 0~50 nm micrfibril w w 3 *T whm crrespndence shuld be addressed. Tel: , Fax: shj@pusan.ac.kr š,, Yung s mdulus ƒ š š q, št, y t t š. BC y ey» ww ƒ üswš (16, 17). w š, w w w w v, drug delivery systems w š. ³ w BC w p w w w p w, A. xylinum w BC œw š ƒƒe ƒe y e v w š (3). Acetbacter ³ e w»- pellicle xk w, pellicle s ƒ w. Williams Cannn (4) A. xylinum BC w w šw y» y s j, e z l s w š w. w A. xylinum x f w» w w ƒ, s œ w š w. e w BC v w z. BC» w w œ v w, w ƒ x ã w š. wr, Acetbacter ³ w w w BC w w cellulse negative mutant (cel - )ƒ w ³ 75

2 76 Jeng-D Kim et al. Kr. J. Micrbil p w w š (3). e w BC y e œv ù x w w» w pellicle xk(7)» ƒ y d š ù e w BC w š. w ƒ š BC w BC y Acetbacter sp. V6 œ ³ w e BC w š, w BC w» z g w. ³ ³ (19) w w m l, Acetbacter sp. V6. ³, BC w» Hestrin Schramm (HS) (8). HS glucse %, yeast extract 0.5%, plypeptne 0.5%, Na H 1H O 0.675% citric acid mnhydrate 0.115% (ph 6). 50 ml HS ƒ w 50 ml cnical flask sqw ³ w w 30 C 7 e w. z x pellicle l s j» w 10 w kw z, ³ w sxk z w (0). sxk 5% (v/v) 50 mlƒ w bttle ( 9cm) w 30 C 7 e w. BC w. BC z w w z, 0.5 N NaOH e w 90 C w s w g. ¾ w z, 105 C w ¾ w d w (5). ³ UV-visible spectrphtmeter (Ultrspec 4000, Pharmacia Bitech, England) w 660 nm Ÿ d w ùkü. BCƒ w hmgenizer 10 q w pellicle ü w s k z, w sxk Ÿ d w (0). BC g/m ùkü. w w x w, Table Figure ùkü s³. BC BC x (JEOL JSM-6390, JEOL TECHNIC LTD., Japan) w. BC pellicle z w.5% glutaraldehyde e w ew. BC pellicle 1 phsphate buffered saline w w z, 70, 80, 90, 100% ethanl ƒƒ 15 w. Isamyl acetate 100% ethanl ƒƒ 0.5:1.5, 1:1, 1.5:0.5 yww 15 w z, 100% isamyl acetate 0 e w. in cating (gld cating) w w. Furier transfrm-infrared spectrscpy (FT-IR) BC pellicle w z, sheet xk w q k 105 C 1 g.» FT-IR Spectrphtmeter (IRAffinity-1, Shimadzu Crp., USA) w 400~4000 cm -1 d w. KBr pwder w q d w. X-ray diffractin (XRD) XRD X-ray Diffractmeter (Rigaku III, Rigaku Crp., Japan) w d w. BC pellicle w sheet xk w q k 105 C, 1 g.» 40 kv, 30 ma Cu Kα radiatin w d θ =5-40, scan speed 10 /min d w. Crystallinity index (Cr.I.) w. CrUIU I am = 100 I 00 I 00» I 00 θ =.8 (00) w z peak (intensity), I am θ =18 z (6). š k BC k BC e w w» w» HS ƒ k.0% ƒw 30 C 7 w Table 1. Glucse, fructse mannitl ƒ BC w Table 1. Effect f carbn surces n cellulse prductin by Acetbacter sp. V6 Carbn surce Cellulse yield (g/m ) Glucse 6.1 Fructse. Lactse. Sucrse.4 Maltse 3.0 Mannitl 1. Varius carbn surces were added at the final cncentratin f % (w/v) t the basic medium cntaining 0.5% yeast extract and 0.5% plypeptne as the nitrgen surce. Cells were cultivated fr 7 days at 30 C. Culture vlume = 50 ml, culture-surface area = 9 cm.

3 Vl. 45, N. 3 Acetbacter sp. w BC p 77 Fig. 1. Effect f glucse cncentratins n cellulse prductin by Acetbacter sp. V6. Cells were cultivated fr 7 days at 30 C in the basic medium. Culture vlume = 50 ml, culture-surface area = 9 cm., p glucse ƒw BC 6.1 g/m ƒ. w e glucse mannitl BC ƒ k Oikawa (13) š w ù, e BC k glucse ew (1). wr, glucse mw BC w v w œw š BC w w mnmer w w š š (), k glucseƒ BC w z q. Glucse k w 7 z BC w w, 3.0% glucse BC (30.0 g/m ) ƒ (Fig. 1). BC BC e w w» w 3.0% glucseƒ w ƒ 0.5% ƒw 30 C 7 w Table. Sytne ƒ ƒ BC (33.3 g/m )ƒ š, Table. Effect f nitrgen surces n cellulse prductin by Acetbacter sp. V6 Nitrgen surce (0.5%) Cellulse yield (g/m ) Yeast extract 6.8 Plypeptne 11.0 Tryptne 16.3 Malt extract 8.0 Crn streep liqur 1. Sytne 33.3 Nne 1.7 Cells were cultivated fr 7 days at 30 C in the medium cntaining glucse with an initial nitrgen surce cncentratin f 0.5% (w/v). Culture vlume = 50 ml, culture-surface area = 9 cm. Fig.. Effect f sytne cncentratins n cellulse prductin by Acetbacter sp. V6. Cells were cultivated in the medium cntaining glucse fr 7 days at 30 C. Culture vlume = 50 ml, culture-surface area = 9 cm. yeast extract BC ùkü. wr, (0) Matsuka (11) e yeast extractƒ ƒ w š šw x w ù, yeast extract sytne w w ƒ, k w BC v. Sytne w 7 w z BC w w, 3.0% sytne BC (39. g/m ) ƒ (Fig. ). BC BC e w w» w Na H 1H O K H 0~1.0% ƒƒ w 30 C 7 w, 0.8% K H BC (41.3 g/m ) ƒ ( ). 0.8% K H ƒ w ƒ NaH KH 0~1.0% ƒƒ w w, ƒ BC w w e w ( ). k BC k BC e w w» w 3% glucse 3% sytne ƒ ƒ» ethanl 0.1% ƒw 30 C 7 w Table 3. Ethanl ƒ BC (43.4 g/m ) ùkü, ù k w ù ethanl û. wr, ethanl s w ATP ƒ jš, pentse phsphate w glucse- 6-phsphate dehydrgenase y ww BC š š (1). Ethanl k w w, 0.4% 44. g/m BC ( ).

4 78 Jeng-D Kim et al. Kr. J. Micrbil Table 3. Effect f secndary substrates n cellulse prductin by Acetbacter sp. V6 Secndary substrate (0.1%) Cellulse yield (g/m ) Glucnic acid 6.8 Lactic acid 35.1 Succinic acid 33.7 Acetic acid 35.0 Fumaric acid 30.3 Frmic acid 36.0 Malic acid 30.7 Citric acid 41.9 Ethanl 43.4 Pyruvic acid 38.6 Nne 30.1 Cells were cultivated in the medium cntaining glucse fr 7 days at 30 C. Culture vlume = 50 ml, culture-surface area = 9 cm. 최적 배지에서 BC 생산 상기 결과에 기초하여 확립된 BC 생산 최적배지 조성은 glucse 3.0%, sytne 3.0%, K HPO 0.8% 및 ethanl 0.4%였 다. 최적 배지조성하에서 회분배양을 실시한 결과, 균체 생육과 BC 생산은 모두 배양시간에 비례하여 증가하였으며, 이에 따라 BC 생산과 균체 생육은 밀접한 관계가 있음을 알 수 있었다. 배 양 8일 후 생성된 BC 생산량은 44.7 g/m 이었다. ph는 배양 경 과에 따라 점차적으로 감소하기 시작하여 배양 8일 경, ph 3.8 로 감소하였다(Fig. 3). 4 Fig. 4. Scanning electrn micrgraph f cellulse prduced by Acetbacter sp. V6. 생산된 BC의 미세구조 최적 배지에서 생산된 BC의 두께를 관찰한 결과, 배양 8일에 약 1 cm 두께의 BC pellicle이 생성되었다(자료 미제시). 이 후, 배양시간을 연장하였으나 pellicle의 두께는 더 이상 증가하지 않 았다. BC의 미세구조를 조사하기 위하여 주사전자현미경을 사용하여 관찰한 결과, 최적 배지에서 생산된 BC는 독특한 미세망상구조 로 이루어져 있었으며, 그 속에 세포가 포획되어 있었다(Fig. 4). 일반적으로 식물성 셀룰로오스는 셀룰로오스 fibre가 평행구조를 이루고 있으며, 미세망상구조는 나타나지 않는다. 따라서 세균유 래 셀룰로오스의 미세망상구조로 인하여 BC가 높은 신장강도, 보수성 등을 가질 수 있는 것으로 추정되었다. FT-IR 일반적으로 식물성 셀룰로오스는 150~1450 cm 영역에서 5 개의 peak 분리를 나타내며, 1000~100 cm 영역에서 4개의 -1-1 Fig. 3. Time curse f cellulse prductin by Acetbacter sp. V6 in an ptimized medium. Cells were cultivated in the ptimized medium fr 8 days at 30 C. Culture vlume = 50 ml, culture-aurface area = 9 cm. Fig. 5. FT-IR spectrum f cellulse prduced by Acetbacter sp. V6.

5 Vl. 45, N. 3 Acetbacter sp. w BC p 79 Fig. 6. X-ray diffractin pattern f cellulse prduced by Acetbacter sp. V6. peak ùkü. w 893 cm -1 CH x w, 1430 cm -1 CH e, 900 cm -1 CH, 3400 cm -1 OH ùkü (18). BC FT-IR spectrum d w, ùkù ƒ ùkû (Fig. 5). z w BC ƒ. XRD Cel I, II, III, IV. Cel I, θ = ƒƒ (101) (101) z peakƒ ùkù, θ =.6 (00) z peak ƒ ùkù (14). BC XRD d w, x Cel I (Fig. 6), Crystallinity index 8% ùkù. š x 1. Byrm, D Micrbil cellulse, pp In D. Byrm (ed.), Bimaterials, Stcktn Press, New Yrk, N.Y., USA.. Cannn, R.E. and S.M. Andersn Bigenesis f bacterial cellulse. Crit. Rev. Micrbil. 17, Czaja, W.K., D.J. Yung, M. Kawecki, and R.M. Brwn, Jr The future prspects f micrbial cellulse in bimedical applicatins. Bimacrml. 8, Dudman, W.F Cellulse prductin by Acetbacter acetigenum and ther Acetbacter spp. J. Gen. Micrbil. 1, Embuscad, M.E., J.N. BeMiller, and J.S. Marks Islatin and partial characterizatin f cellulse prduced by Acetbacter xylinum. Fd Hydrcll. 10, Fcher, B., M.T. Palma, M. Canetti, G. Trri, C. Csentin, and G. Gastaldi Structural differences between nn-wd plant cellulses: evidence frm slid state NMR, vibratinal spectrscpy and X-ray diffractmetry. Ind. Crps Prd. 13, Fntana, J.D., A.M. De Suza, C.K. Fntana, I.L. Trriani, J.C. Mreschi, B.J. Galltti, S.J. De Suza, G.P. Narcis, J.A. Bichara, and L.F.X. Farah Acetbacter cellulse pellicle as a temprary skin substitute. Appl. Bichem. Bitechnl. 4/5, Hestrin, S. and M. Schramm Synthesis f cellulse by Acetbacter xylinum. Bichem. J. 58, Klemm, D., D. Schumann, U. Udhard, and S. Marsch Bacterial synthesized cellulse - artficial bld vessels fr micrsurgery. Prg. Plym. Sci. 6, K, J.Y., K.S. Shin, B.D. Yn, and W.Y. Chi. 00. Prductin f baterial cellulse by Axetbacter xylinum GS11. Kr. J. Appl. Micrbil. Bitechnl. 30, Matsuka, M., T. Tsuchida, K. Matsushita, O. Adachi, and F. Yshinaga A synthetic medium fr bacterial cellulse prductin by Acetbacter xylinum subsp. sucrfermentans. Bisci. Bitech. Bichem. 60, Naritmi, T., T. Kuda, H. Yan, and F. Yshinaga Effect f ethanl n bacterial cellulse prductin frm fructse in cntinuus culture. J. Ferment. Bieng. 85, Oikawa T., T. Ohtti, and M. Ameyama Prductin f cellulse frm D-mannitl by Acetbacter xylinum KU-1. Bisci. Bitech. Bichem. 59, Paek, H.S., J.S. Park, S.M. J, W.S. Lee, and K.J. Kim The effects f preparatin cnditins f celluse/n-methylmrphline- N-xide slutins n physical prperties f fibers. J. Krean Fiber Sc. 30, Rainer, J. and F.F. Luiz Prductin and applicatin f micrbial cellulse. Plym. Degrad. Stab. 58, Rss, P., H. Weinhuse, Y. Alni, D. Michaeli, P. Weinberger- Ohana, R. Mayer, S. Braun, E. de Vrm, G.A. van der Marel, J.H. van Bm, and M. Benziman Regulatin f cellulse synthesis in Acetbacter xylinum by cyclic diguanylic acid. Nature 35, Rss, P., Y. Alni, H. Weinhuse, D. Michaeli, P. Weinberger- Ohana. R. Mayer, and M. Benziman Cntrl f cellulse synthesis in Acetbacter xylinum. A unique guanyl lignucletide is the immediate activatr f the cellulse synthase. Carbhyd. Res. 149, Sakran, M.A Mechanism f cellulse plymer reactins with sme catin-exchanged mntmrillnite catalysts. J. Radianal. Nucl. Chem. 13, Sn, H.J., O.M. Lee, Y.G. Kim, and S.J. Lee Islatin and identificatin f cellulse-prducing bacteria. Kr. J. Appl. Micrbil. Bitechnl. 8, Sn, H.J., O.M. Lee, Y.G. Kim, Y.K. Park, and S.J. Lee Characteristics f cellulse prductin by Acetbacter sp. A9 in static culture. Krean J. Bitechnl. Bieng. 15, Sutherland, I.W Nvel and estabilished applicatins f micrbial plysaccharides. Tibtech. 16, Tda, K., T. Asakura, M. Fukaya, E. Entani, and Y. Kawamura Cellulse prductin by acetic acid-resistant Acetbacter xylinum. J. Ferment. Bieng. 84, Valla, S. and J. Kjsbakken Cellulse negative mutants f Acetbacter xylinum. J. Gen. Micrbil. 18, Williams, W.S. and R.E. Cannn Alternative envirnmental rles fr cellulse prduced by Acetbacter xylinum. Appl. Envirn. Micrbil. 55, Yamanaka, S. and K. Watanabe Applicatins f bacterial cellulse in cellulsic plymers, pp In R. Gillbert (ed.),

6 80 Jeng-D Kim et al. Kr. J. Micrbil Cellulsic plymers - Blends and cmpsites, Hanser Inc., Cincinnati, OH, USA. 6. Yamanaka, S., K. Watanabe, N. Kitamura, M. Iguchi, S. Mitsuhashi, Y. Nishi, and M. Uryu The structure and mechanical prperties f sheets prepared frm bacterial cellulse. J. Mat. Sci. 4, (Received August 8, 009/Accepted September, 009) ABSTRACT : Prductin and Structural Analysis f Cellulse by Acetbacter sp. V6 Using Static Culture Jeng-D Kim, H-Il Jung, Jin-Ha Jeng, Ki-Hyun Park, Yung-Dng Jen, Dae-Yun Hwang, Chung-Yel Lee, and Hng-J Sn* (Cllege f Natural Resurces and Life Science, Pusan Natinal University, Miryang , Republic f Krea) The ptimal medium cmpsitins fr the prductin f bacterial cellulse (BC) by a Acetbacter sp. V6, which was islated frm the traditinally fermented vinegar in Krea, were investigated in static cultures. The ptimum medium cmpsitins fr BC prductin were 3% glucse, 3% sytne, 0.8% K H, and 0.4% ethanl, respectively. Adding NaH r KH had nt shwn the increase in BC prductin. Under the ptimum medium cmpsitins, the highest BC prductin was g/m in 8 days and the thickness f BC pellicle was abut 1 cm. Structural prperties f BC prduced in the ptimal medium were studied using Furier-transfrm infrared spectrscpy and X-ray diffractmeter. BC frm the ptimal medium was fund t be f cellulse type I, the same as typical native cellulse. N difference in the cmpsitins between bacterial and plant cellulses, but BC shwed unique micr-netwrk structure and high crystallinity (8%).