, Volume 20, Issue 6, pp 2971–2979 | Cite as

One-step production of nanofibrillated bacterial cellulose (NFBC) from waste glycerol using Gluconacetobacter intermedius NEDO-01

  • Ryota Kose
  • Naoki Sunagawa
  • Makoto Yoshida
  • Kenji Tajima
Original Paper


A major by-product of biodiesel production is waste glycerol, which has numerous potential applications. In this study, we isolated a novel bacterium capable of producing cellulose from waste glycerol, and identified it as a novel strain (named NEDO-01) of Gluconacetobacter intermedius. Scanning electron microscopy revealed that the morphology of the pellicle produced by NEDO-01 was similar to that of cellulose produced by Gluconacetobacter hansenii ATCC23769. Furthermore, X-ray diffraction and solid-state nuclear magnetic resonance spectroscopic analyses suggested that cellulose produced by NEDO-01 had molecular and crystalline structures similar to those of cellulose produced by ATCC23769. After the optimization of cultivation conditions, NEDO-01 mediated the one-step production of nanofibrillated bacterial cellulose (NFBC) from waste glycerol in a medium supplemented with carboxymethyl cellulose. Transmission electron microscopic analysis revealed that the NFBC was composed of relatively uniform fibers with diameters of approximately 20 nm. NFBC was produced as uniform water suspensions, the yield of which was 3.4 g/L from cultivation in 7.5 L medium in a 10-L jar fermenter. The bioconversion of waste glycerol to NFBC, which has superior fluidity, moldability, and miscibility, has a wide variety of applications, including potential uses in the medical and materials engineering fields.


Fibrillated cellulose nanofiber Biodiesel fuel by-product (BDF-B) Acetobacter xylinum Bioconversion Biorefinery Carboxymethyl cellulose 



Waste glycerol was a kind gift from Hokusei Kigyo Co. Ltd. This work was supported by a Grant for Advanced Industrial Technology Development in 2011 (11B12009) from the New Energy and Industrial Technology Development Organization (NEDO) of Japan. We thank Mr. Kenji Ohkubo of Hokkaido University for his technical support in TEM and SEM observations. A part of this work was conducted at Hokkaido University, supported by “Nanotechnology Platform” Program of the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan.


  1. Abdul Khalil HPS, Bhat AH, Ireana Yusra AF (2012) Green composites from sustainable cellulose nanofibrils: a review. Carbohydr Polym 87:963–979CrossRefGoogle Scholar
  2. Abe K, Yano H (2009) Comparison of the characteristics of cellulose microfibril aggregates of wood, rice straw and potato tuber. Cellulose 16:1017–1023CrossRefGoogle Scholar
  3. Brown RM Jr (1996) The biosynthesis of cellulose. J Macromol Sci A 33:1345–1373CrossRefGoogle Scholar
  4. Chen H–H, Chen L-C, Huang H-C, Lin S-B (2011) In situ modification of bacterial cellulose nanostructure by adding CMC during the growth of Gluconacetobacter xylinus. Cellulose 18:1573–1583CrossRefGoogle Scholar
  5. Cheng K-C, Catchmark JM, Demirci A (2009) Effect of different additives on bacterial cellulose production by Acetobacter xylinum and analysis of material property. Cellulose 16:1033–1045CrossRefGoogle Scholar
  6. Cheng K-C, Catchmark JM, Demirci A (2011) Effects of CMC addition on bacterial cellulose production in a biofilm reactor and its paper sheets analysis. Biomacromolecules 12:730–736CrossRefGoogle Scholar
  7. Chi Z, Pyle D, Wen Z, Frear C, Chen S (2007) A laboratory study of producing docosahexaenoic acid from biodiesel-waste glycerol by microalgal fermentation. Process Biochem 42:1537–1545CrossRefGoogle Scholar
  8. Czaja W, Krystynowicz A, Bielecki S, Brown RM Jr (2006) Microbial cellulose—the natural power to heal wounds. Biomaterials 27:145–151CrossRefGoogle Scholar
  9. da Silva GP, Mack M, Contiero J (2009) Glycerol: a promising and abundant carbon source for industrial microbiology. Biotechnol Adv 27:30–39CrossRefGoogle Scholar
  10. Doblin MS, Kurek I, Jacob-wilk D, Delmer DP (2002) Cellulose biosynthesis in plants: from genes to rosettes. Plant Cell Physiol 43:1407–1420CrossRefGoogle Scholar
  11. Haigler CH, White AR, Brown RM Jr, Cooper KM (1982) Alteration of in vivo cellulose ribbon assembly by carboxymethylcellulose and other cellulose derivatives. J Cell Biol 94:64–69CrossRefGoogle Scholar
  12. Henriksson M, Henriksson G, Berglund LA, Lindström T (2007) An environmentally friendly method for enzyme-assisted preparation of microfibrillated cellulose (MFC) nanofibers. Eur Polym J 43:3434–3441CrossRefGoogle Scholar
  13. 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
  14. Hirai A, Tsuji M, Yamamoto H, Horii F (1998) In situ crystallization of bacterial cellulose III. Influences of different polymeric additives on the formation of microfibrils as revealed by transmission electron microscopy. Cellulose 5:201–213CrossRefGoogle Scholar
  15. Isogai A, Saito T, Fukuzumi H (2011) TEMPO-oxidized cellulose nanofibers. Nanoscale 3:71–85CrossRefGoogle Scholar
  16. Kataoka Y, Kondo T (1999) Quantitative analysis for the cellulose Iα crystalline phase in developing wood cell walls. Int J Biol Macromol 24:37–41CrossRefGoogle Scholar
  17. Kim U-J, Eom SH, Wada M (2010) Thermal decomposition of native cellulose: influence on crystallite size. Polym Degrad Stab 95:778–781CrossRefGoogle Scholar
  18. Kimura S, Kondo T (2002) Recent progress in cellulose biosynthesis. J Plant Res 115:297–302CrossRefGoogle Scholar
  19. Kouda T, Yano H, Yoshinaga F (1997) Effect of agitator configuration on bacterial cellulose productivity in aerated and agitated culture. J Ferment Bioeng 83:371–376CrossRefGoogle Scholar
  20. Mansikkamäki P, Lahtinen M, Rissanen K (2005) Structural changes of cellulose crystallites induced by mercerisation in different solvent systems; determined by powder X-ray diffraction method. Cellulose 12:233–242CrossRefGoogle Scholar
  21. Nogi M, Yano H (2008) Transparent nanocomposites based on cellulose produced by bacteria offer potential innovation in the electronics device industry. Adv Mater 20:1849–1852CrossRefGoogle Scholar
  22. Pei A, Butchosa N, Berglund LA, Zhou Q (2013) Surface quaternized cellulose nanofibrils with high water absorbency and adsorption capacity for anionic dyes. Soft Matter 9:2047–2055CrossRefGoogle Scholar
  23. Ross P, Mayer R, Benziman M (1991) Cellulose biosynthesis and function in bacteria. Microbiol Rev 55:35–58Google Scholar
  24. Saito T, Nishiyama Y, Putaux J-L, Vignon M, Isogai A (2006) Homogeneous suspensions of individualized microfibrils from TEMPO-catalyzed oxidation of native cellulose. Biomacromolecules 7:1687–1691CrossRefGoogle Scholar
  25. Saxena IM, Brown RM Jr (2005) Cellulose biosynthesis: current views and evolving concepts. Ann Bot Lond 96:9–21CrossRefGoogle Scholar
  26. Taylor NG (2008) Cellulose biosynthesis and deposition in higher plants. New Phytol 178:239–252CrossRefGoogle Scholar
  27. Toda K, Yamamoto H, Yoshida M (2013) Crystallization of cellulose microfibrils in wood cell wall by repeated dry-and-wet treatment, using X-ray diffraction technique. Cellulose 20:633–643CrossRefGoogle Scholar
  28. Wågberg L, Decher G, Norgren M, Lindström T, Ankerfors M, Axnäs K (2008) The build-up of polyelectrolyte multilayers of microfibrillated cellulose and cationic polyelectrolytes. Langmuir 24:748–795CrossRefGoogle Scholar
  29. Yamamoto H, Horii F (1993) CP/MAS 13C NMR analysis of the crystal transformation induced for valonia cellulose by annealing at high temperatures. Macromolecules 26:1313–1317CrossRefGoogle Scholar
  30. Yamamoto H, Horii F, Hirai A (1996) In situ crystallization of bacterial cellulose II. Influences of different polymeric additives on the formation of celluloses Iα and Iβ at the early stage of incubation. Cellulose 3:229–242CrossRefGoogle Scholar
  31. Yamanaka S, Watanabe K, Kitamura N, Iguchi M, Mitsuhashi S, Nishi Y, Uryu M (1989) The structure and mechanical properties of sheets prepared from bacterial cellulose. J Mater Sci 24:3141–3145CrossRefGoogle Scholar
  32. Yoshinaga F, Tonouchi N, Watanabe K (1997) Research progress in production of bacterial cellulose by aeration and agitation culture and its application as a new industrial material. Biosci Biotech Biochem 61:219–224CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2013

Authors and Affiliations

  • Ryota Kose
    • 1
    • 3
  • Naoki Sunagawa
    • 2
    • 4
  • Makoto Yoshida
    • 1
  • Kenji Tajima
    • 1
    • 2
  1. 1.Faculty of EngineeringHokkaido UniversitySapporoJapan
  2. 2.Graduate School of Chemical Sciences and EngineeringHokkaido UniversitySapporoJapan
  3. 3.Faculty of AgricultureTokyo University of Agriculture and TechnologyFuchu-shiJapan
  4. 4.Department of Biomaterial Sciences, Graduate School of Agricultural and Life SciencesThe University of TokyoBunkyo-kuJapan

Personalised recommendations