, Volume 17, Issue 1, pp 139–151 | Cite as

Structure elucidation of uniformly 13C-labeled bacterial celluloses from different Gluconacetobacter xylinus strains

  • Stephanie Hesse-Ertelt
  • Thomas Heinze
  • Eiji Togawa
  • Tetsuo Kondo


The morphological and supramolecular structures of native cellulose pellicles from two strains of Gluconacetobacter xylinus (ATCC 53582, ATCC 23769) were investigated. Samples had been statically cultivated in Hestrin-Schramm medium containing fully 13C-labeled β-d-glucose-U-13C6 as the sole source of carbon. The results are compared with structure data of bacterial celluloses with a natural 13C abundance of 1.1%. Non-enriched and 13C-labeled cellulose pellicles formed crystalline structures as revealed by cross-polarized/magic-angle spinning (CP/MAS) 13C{1H}-NMR and near infrared (NIR) FT-Raman spectroscopic measurements as well as wide-angle X-ray diffraction (WAXD) investigations. Atomic force microscopy (AFM) was applied for analyzing fiber morphologies and surface properties. For the first time, details about the manipulation of fiber widths and pellicle formation were shown for different bacterial strains of G. xylinus depending on the use of β-d-glucose-U-13C6 for the biosynthesis.


Bacterial cellulose Gluconacetobacter xylinus 13C-Labeling Biosynthesis 13C Nuclear magnetic resonance NIR FT-Raman Atomic force microscopy Wide-angle X-ray diffraction Crystallinity 


A. xylinum

Acetobacter xylinum


Atomic force microscopy


American type culture collection


Bacterial strain ATCC 23769


Bacterial cellulose


Crystallinity index


Cross polarization


Deutsche Sammlung von Mikroorganismen


Fourier transformed Raman


Full width at half maximum

G. xylinus

Gluconacetobacter xylinus



Iα, Iβ

Cellulose modifications


Crystallinity value obtained by NMR


Incredible natural abundance double quantum transfer experiment


Magic angle spinning


Near infrared


Nuclear magnetic resonance


Nematic ordered cellulose


Bacterial strain ATCC 53582


Two pulse phase modulation


Wide-angle X-ray diffraction


Crystallinity value obtained by WAXD



This research was financial supported by the Friedrich Schiller University of Jena (Foerderung von Frauen in Forschung und Lehre, Kapitel 1524/TG 84, 2002) for StHE, by MAFF Nanotechnology Project, Ministry of Agriculture, Forestry and Fisheries, and partly by a Grant-in-Aid for Scientific Research (No. 14360101), Japan Society for the Promotion of Science (JSPS) for TK. The authors are also indebted to Dr. U. Sternberg (FZ Karlsruhe, Germany) for partly financing d-glucose-U-13C6. We thank Dr. S. Kimura and Ms. A. Morohoshi (FFPRI Tsukuba, Japan) for their kind assistance through this research, and Dr. W. Plass and Dr. A. Pohlmann (IAAC, FSU Jena, Germany) for providing the NIR FT-Raman spectrometer. StHE particularly thanks TK for the chance of sample preparation and characterization with his former group at the Forestry and Forest Products Research Institute (FFPRI), Matusnosato 1, Tsukuba, Ibaraki 305-8687, Japan.


  1. Arashida T, Ishino T, Kai A, Hatanaka K, Akaike T, Matsuzaki K, Kaneko Y, Mimura T (1993) Biosynthesis of cellulose from culture media containing 13C-labeled glucose as a carbon source. J Carbohydr Chem 12:641–649CrossRefGoogle Scholar
  2. Atalla RH (1976) Raman spectral studies of polymorphy in cellulose. Part I: Celluloses I and II. Appl Polym Symp 28:659–669Google Scholar
  3. Atalla RH, VanderHart DL (1999) The role of solid state 13C NMR spectroscopy in studies of the nature of native celluloses. Solid State Nucl Magn Reson 15:1–19CrossRefGoogle Scholar
  4. Blackwell J, Vasko PD, Koenig JL (1970) Infrared and Raman spectra of the cellulose from the cell wall of Valonia ventricosa. J Appl Phys 41:4375–4379CrossRefGoogle Scholar
  5. Bohn A (2000) Röntgenuntersuchungen zur Vorzugsorientierung und übermolekularen Struktur nativer und regenerierter Cellulose. Dissertation, TU BerlinGoogle Scholar
  6. Bohn A, Fink HP, Ganster J, Pinnow M (2000) X-ray texture investigations of bacterial cellulose. Macromol Chem Phys 201:1913–1921CrossRefGoogle Scholar
  7. Brown RM Jr (1989) Cellulose biogenesis and a decade of progress: a personal perspective. In: Schuerch C (ed) Cellulose and wood: chemistry and technology. John Wiley and Sons, New York, pp 639–657Google Scholar
  8. Brown RM Jr, Willison JHM, Richardson CL (1976) Cellulose biosynthesis in Acetobacter xylinum: visualization of the site of synthesis and direct measurement of the in vivo process. Proc Natl Acad Sci USA 73:4565–4569CrossRefGoogle Scholar
  9. Earl WL, VanderHart DL (1982) Measurement of 13C chemical shifts in solids. J Magn Reson 48:35–54Google Scholar
  10. Erata T, Shikano T, Yunoki S, Takai M (1997) The complete assignment of the 13C CP/MAS NMR spectrum of native cellulose by using 13C labeled glucose. Cellulose Commun 4:128–131Google Scholar
  11. Evans RJ, Wang D, Agblevor FA, Chum HL, Baldwin SD (1996) Mass spectrometric studies of the thermal decomposition of carbohydrates using 13C-labeled cellulose and glucose. Carbohydr Res 281(2):219–235CrossRefGoogle Scholar
  12. Fink HP, Walenta E (1994) Models of cellulose structure from the viewpoint of the cellulose I → II transition. Papier 12:739–748Google Scholar
  13. Fink HP, Hofmann D, Philipp B (1995) Some aspects of lateral chain order in cellulosics from X-ray scattering. Cellulose 2:51–70Google Scholar
  14. Fink HP, Purz HJ, Bohn A, Kunze J (1997) Investigation of the supramolecular structure of never dried bacterial cellulose. Macromol Symp 120:207–217Google Scholar
  15. Fischer S, Schenzel K, Fischer K, Diepenbrock W (2005) Applications of FT Raman spectroscopy and micro spectroscopy characterizing cellulose and cellulosic biomaterials. Macromol Symp 223:41–56CrossRefGoogle Scholar
  16. Gagnaire D, Taravel FR (1980) Biosynthèse de cellulose bactérienne à partir de d-glucose uniformément enrichi en 13C. Eur Biochem 103:133–143CrossRefGoogle Scholar
  17. Ganster J, Fink HP (1999) Physical constants of cellulose. In: Immergut EH, Grulke EA (eds) Polymer handbook, 4th edn. Wiley, New York, p V/135ffGoogle Scholar
  18. Groebe A, Chmiel H, Strathmann H (1991) Verfahren zur Herstellung von Cellulose-Membranen aus bakteriell erzeugter Cellulose. EU Patent No. 0416470A2Google Scholar
  19. Hermans PH, Weidinger A (1946) On the recrystallization of amorphous cellulose. J Am Chem Soc 68:2547–2552CrossRefGoogle Scholar
  20. Hesse S, Jaeger C (2005) Determination of the 13C chemical shift anisotropies of cellulose I and cellulose II. Cellulose 12:5–14CrossRefGoogle Scholar
  21. Hesse S, Kondo T (2005) Behavior of cellulose production of Acetobacter xylinum in 13C-enriched cultivation media including movements on nematic ordered cellulose templates. Carbohydr Polym 60(4):457–465CrossRefGoogle Scholar
  22. Hesse-Ertelt S, Witter R, Ulrich AS, Kondo T, Heinze T (2008) Spectral assignments and anisotropy data of cellulose Iα: 13C-NMR chemical shift data of cellulose Iα determined by INADEQUATE and RAI techniques applied to uniformly 13C-labeled bacterial celluloses of different Gluconacetobacter xylinus strains. Magn Reson Chem 46:1030–1036CrossRefGoogle Scholar
  23. Hestrin S, Schramm M (1954) Synthesis of cellulose by Acetobacter xylinum. 2. Preparation of freeze dried cells capable of polymerizing glucose to cellulose. Biochem J 58:345–352Google Scholar
  24. Hirai A, Tsuji M, Horii F (1997) Culture conditions producing structure entities composed of cellulose I and II in bacterial cellulose. Cellulose 4:239–245CrossRefGoogle Scholar
  25. Jayme G, Knolle H (1964) The empirical X-ray determination of the degree of crystallinity of cellulosic material. Papier 18:249–255Google Scholar
  26. Kai A, Arashida T, Hatanaka K, Akaike T, Matsuzaki K, Mimura T, Kaneko Y (1994) Analysis of the biosynthetic process of cellulose and curdlan using 13C-labeled glucose. Carbohydr Polym 23:235–239CrossRefGoogle Scholar
  27. Kai A, Karasawa H, Kikawa M, Hatanaka K, Matsuzaki K, Mimura T, Kaneko Y (1998) Biosynthesis of 13C-labeled branched polysaccharides by pestalotiopsis from 13C-labeled glucoses and the mechanism of formation. Carbohydr Polym 35:271–278CrossRefGoogle Scholar
  28. Kast W, Flaschner L (1948) Eine röntgenographische Methode zur Bestimmung des Verhältnisses von kristalliner und amorpher Substanz in Zellulosefasern. Coll Polym Sci 111(1):6–15Google Scholar
  29. Klemm D, Schumann D, Udhardt U, Marsch S (2001) Bacterial synthesized cellulose—artificial blood vessels for microsurgery. Prog Polym Sci 26:1561–1603CrossRefGoogle Scholar
  30. Klemm D, Heublein B, Fink HP, Bohn A (2005) Cellulose: Fascinating biopolymer and sustainable raw material. Angew Chem Int Ed 44:3358–3393CrossRefGoogle Scholar
  31. Kondo T, Togawa E, Brown RM Jr (2001) Nematic ordered cellulose: a concept of glucan chain association. Biomacromolecules 2:1324–1330CrossRefGoogle Scholar
  32. Kondo T, Nojiri M, Hishikawa Y, Togawa E, Romanovicz D, Brown RM Jr (2002) Biodirected epitaxial nanodeposition of polymers on oriented macromolecular templates. Proc Natl Acad Sci USA 99:14008–14013CrossRefGoogle Scholar
  33. Kono H, Numata Y (2006) Structural investigation of cellulose Iα and Iβ by 2D RFDR NMR spectroscopy: determination of sequence of magnetically inequivalent d-glucose units along cellulose chain. Cellulose 13:317–326CrossRefGoogle Scholar
  34. Kono H, Yunoki S, Shikano T, Fujiwara M, Erata T, Takai MJ (2002) CP/MAS 13C NMR study of cellulose and cellulose derivatives. 1. Complete assignment of the CP/MAS 13C NMR spectrum of the native cellulose. Am Chem Soc 124:7506–7511CrossRefGoogle Scholar
  35. Kono H, Erata T, Takai M (2003) Determination of the through-bond carbon–carbon and carbon-proton connectivities of the native celluloses in the solid state. Macromolecules 36:5131–5138CrossRefGoogle Scholar
  36. Kuga S, Brown RM Jr (1991) Physical structure of cellulose microfibrils: Implication for biogenesis. In: Haigler CH, Weimer PJ (eds) Biosynthesis and biodegradation of cellulose. M. Dekker, Inc, New York, Basel, Hong Kong, p VI/125ffGoogle Scholar
  37. Kuga S, Takagi S, Brown RM Jr (1993) Native folded-chain cellulose II. Polymer 34:3293–3297CrossRefGoogle Scholar
  38. Meyer KH, Misch L (1937) Position des atomes dans le nouveau module spatial de la cellulose. Helv chim Acta 20:232–244CrossRefGoogle Scholar
  39. Numata Y, Kono H, Kawano S, Erata T, Takai M (2003) Cross-polarization/magic-angle spinning 13C nuclear magnetic resonance study of cellulose I–ethylenediamine complex. J Biosci Bioeng 96:461–466Google Scholar
  40. Sarko A, Muggli R (1974) Packing analysis of carbohydrates and polysaccharides. III. Valonia cellulose and cellulose II. Macromolecules 7:486–494CrossRefGoogle Scholar
  41. Schenzel K, Fischer S (2001) NIR FT Raman spectroscopy–a rapid analytical tool for detecting the transformation of cellulose polymorphs. Cellulose 8:49–57CrossRefGoogle Scholar
  42. Schenzel K, Fischer S, Brendler E (2005) New method for determining the degree of cellulose I crystallinity by means of FT Raman spectroscopy. Cellulose 12:223–231CrossRefGoogle Scholar
  43. Seifert M, Hesse S, Kabrelian V, Klemm D (2004) Controlling the water content of never dried and reswollen bacterial cellulose by the addition of water-soluble polymers to the culture medium. J Polym Sci A (Chem) 42:463–470CrossRefGoogle Scholar
  44. Togawa E, Kondo T (1999) Change of morphological properties in drawing water-swollen cellulose films prepared from organic solutions: a view of molecular orientation in the drawing process. J Polym Sci B (Phys) 37:451–459CrossRefGoogle Scholar
  45. Udhardt U, Hesse S, Klemm D (2005) Analytical investigations of bacterial cellulose. Macromol Symp 223:201–212CrossRefGoogle Scholar
  46. VanderHart DL, Atalla RH (1984) Studies of microstructure in native celluloses using solid state 13C NMR. Macromolecules 17:1465–1472CrossRefGoogle Scholar
  47. Watanabe K, Tabuchi M, Morinaga Y, Yoshinaga F (1998) Structural features and properties of bacterial cellulose produced in agitated culture. Cellulose 5:187–200CrossRefGoogle Scholar
  48. Wiley JH, Atalla RH (1987) Bands assignments in the Raman spectra of celluloses. Carbohydr Res 160:113–129CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2009

Authors and Affiliations

  • Stephanie Hesse-Ertelt
    • 1
  • Thomas Heinze
    • 1
  • Eiji Togawa
    • 2
  • Tetsuo Kondo
    • 3
  1. 1.Friedrich Schiller University of JenaCentre of Excellence for Polysaccharide ResearchJenaGermany
  2. 2.Forestry and Forest Products Research Institute (FFPRI)Tsukuba, IbarakiJapan
  3. 3.Bio-Architecture Center (KBAC) and Graduate School of Bioresource and Bioenvironmental SciencesKyushu UniversityFukuokaJapan

Personalised recommendations