Microbial Reduction of Graphene Oxide by ‎Lactobacillus Plantarum

Document Type : Research Paper


1 Genetic Engineering and Biotechnology Institute, Marmara Research Centre, TUBİTAK, Kocaeli, TURKEY.

2 ‎Food Institute, Marmara Research Center, TUBITAK, Kocaeli, Turkey.‎

3 ‎Materials Institute, Marmara Research Center, TUBITAK, Kocaeli, Turkey.‎

4 ‎Genetic Engineering and Biotechnology Institute, TUBITAK, Marmara Research Center, ‎Kocaeli, Turkey.‎


   Here, we report that the reduced graphene oxide nanosheets were successfully synthesized using the ‎Lactobacillus plantarum biomass in a simple, environmentally friendly and scalable manner. We ‎produced graphene oxide by oxidization and exfoliation of graphite flakes with modified Hummer's ‎method and then reduced to reduced graphene oxide by using Lactobacillus plantarum biomass as a ‎reducing agent. Samples were characterized using Fourier transform infrared spectroscopy, X-ray ‎photoelectron spectroscopy, transmission electron microscopy, scanning electron microscopy, ‎microconfocal raman spectroscopy and thermogravimetric analysis. After the reduction, we observed ‎that a considerable decrease in the oxygen containing functional groups of graphene oxide and an ‎increase in C/O ratio from 1.7 to 3.3 in which confirms sp2 graphitic carbons increase. Mainly, we ‎observed a significant decrease in epoxy and alkoxy functionalities. Furthermore, we determined an ‎exfoliation of graphene oxide to one or several (2-5) layers after the complete reduction. In addition to ‎reducing potential, Lactobacillus plantarum biomass also plays an important role as stabilizing agent; ‎here the reduced graphene oxide showed a good stability in water. The green synthesis reported in this ‎work is concerned with the production of high purity water-dispersible reduced graphene oxide using ‎Lactobacillus plantarum CCM 1904.‎


  1. Lee, C. G., Wei, X. D., Kysa,r J. W., Hone, J., (2008). “Measurement of the elastic properties and intrinsic     strength of monolayer graphene”, Science, 321: 385-388.
  2. Balandin, A. A., Ghosh, S., Bao, W., Calizo, I., Teweldebrhan, D., Miao, F., Lau, C. N., (2008). “Superior thermal conductivity of single-layer graphene”, Nano Letters, 8: 902-907.
  3. Orlita, M., Faugeras, C., Plochocka, P., Neugebauer, P., Martinez, G., Maude, D. K., Barra, A. L., Sprinkle, M., Berger, C., De Heer, W. A., Potemski, M., (2008). “Approaching the dirac point in high mobility multilayer epitaxial graphene”, Physical Review Letter, 101: 267601.
  4. Cai, W., Zhu, Y., Li, X., Piner, R. D., Ruoff, R. S., (2009). “Large area few-layer graphene/graphite films as transparent thin conducting electrodes”, Applied Physical Letters, 95: 123115.
  5. Li, X., Zhu, Y., Cai, W., Borysiak, M., Han, B., Chen, D., Piner, R. D., Colombo, L., Ruoff, R. S., (2009). “Transfer of large-area graphene films for high-performance transparent conductive electrodes”, Nano Letters, 9: 4359-4363.
  6. Geim, K. A., Novoselov, K. S., (2007).  “The rise of graphene”, Nature Materials, 6: 183-191.
  7. Liu, Z., Lau, S. P., Yan, F., (2015). “Functionalized graphene and other two-dimensional materials for photovoltaic devices: device design and processing”, Chemical Society Reviews, 44: 5638-5679.
  8. Xia, F., Mueller, T., Lin, Y. M., Valdes-Garcia A., Avouris, P., (2009). “Ultrafast graphene photodetector”, Nature Nanotechnology, 4: 839-843.
  9. Liu, M., Zhang, R., Chen, W., (2014). “Graphene-supported nanoelectrocatalysts for fuel cells: synthesis, properties, and applications”, Chemical Reviews, 114: 5117-5160.
  10. Wu, Z. S., Feng, X., Cheng, H. M., (2013). “Recent advances in graphene-based planar micro-supercapacitors for on-chip energy storage”, National Science Reviews, 1: 277-292.
  11. Zhai, Y., Dou, Y., Zhao, D., Fulvio, F. P., Mayes, R. T., Dai, S., (2011). “Carbon materials for chemical capacitive energy storage”, Advanced Materials, 23: 4828-4850.
  12. Liu, Y., Dong, X., Chen, P., (2012). “Biological and chemical sensors based on graphene materials”, Chemical Society Reviews, 4: 2283-2307.
  13. Mao, H. Y., Laurent, S., Chen, W., Akhavan, O., Imani, M., Ashkarran, A. A., Mahmoudi M., (2013). “Graphene: promises, facts, opportunities, and challenges in nanomedicine”, Chemical Reviews, 113: 3407-3424.
  14. Kumar, S., Yadav, R. K., Ram, K., Aguiar, A., Koh, J., Sobral, A. J. F. N., (2018). “Graphene oxide modified cobalt metallated porphyrin photocatalyst for conversion of formic acid from carbon dioxide”, Journal of CO2 Utilization, 27: 107-114.
  15. Kumar, S., Koh, J., (2014). “Physiochemical and optical properties of chitosan based graphene oxide bionanocomposite”, International Journal of Biological Macromolecules, 70: 559-564.
  16. Raccichini, R., Varzi, A., Passerini, S., Scrosati, B., (2015). “The role of graphene for electrochemical energy storage”, Nature Materials, 14: 271-279.
  17. Azamat, J., (2018). “Application of Functionalized Graphene Oxide Nanosheet in Gas Separation”, International Journal of Nanoscience and Nanotechnoogy, 14: 165-175.
  18. Kumar, V., Khandelval, G., (2016). “Graphene-based Flexible and Stretchable Bioelectronics in Health Care Systems”, Journal of Analytical and Pharmaceutical Research, 3: 53-55.
  19. Roy-Mayhew, J. D., Aksay, I. A., (2014). “Dye-Sensitized Solar Cells”, Chemical Reviews, 114: 6323-6348.
  20. Smith,,A.D., Elgammal,,K., Niklaus, F., Delin, A., Fischer, A. C., Vaziri, S., Forsberg, F., Rasander, M., Hugosson, H., Berqvist, L., Schröder, S., Kataria, S., Östling, M., Lemme, M. C., (2015). “Resistive graphene humidity sensors with rapid and direct electrical readout”, Nanoscale, 7: 1909919109.
  21. Geim, A. K., (2009). “Graphene: status and prospects”, Science, 324: 1530-1534.
  22. Novoselov, K. S., Geim, A. K., Morozov, S. V., Jiang, D., Zhang, Y., Dubonos, S. V., Grigorieva, I. V., Firsov, A. A., (2004). “Electric field effect in atomically thin carbon films”, Science, 306: 666-669.
  23. Berger, C., Song, Z., Li, X., Wu, X., Brown, N., Naud, C., Mayou, D., Li, T., Hass, J., Marchenkov, A. N., Conrad, E. H., First, P. N., De Heer, W. A., (2006). “Electronic confinement and coherence in patterned epitaxial graphene”, Science, 312: 1191-1196.
  24. Wintterlin, J., Bocquet, M. L., (2009). “Graphene on metal surface”, Surface Science, 603: 1841-1852.
  25. Land, T. A., Michely, T., Behm, R. J., Hemminger, J. C., Comsa, G., (1992). “STM investigation of single layer graphite structures produced on Pt (111) by hydrocarbon decomposition”, Surface Science, 264: 261-270.
  26. Eizenberg, M., Blakely, J. M, (1979). “Carbon monolayer phase condensation on Ni (111)”, Surface Science, 82: 228-236.
  27. Kim, K. S., Zhao, Y., Jang, H., Lee, S. Y., Kim, J. M., Kim, K. S, Ahn, J. H., Kim, P., Choi, J. Y., Hong, B. H., (2009). “Large scale pattern growth of graphene films for stretchable transparent electrodes”, Nature, 457: 706-710.
  28. Sakamoto, J., Van Heijst, J., Lukin, O., Schluter, A. D., (2009). “Two dimensional polymers: just a dream of synthetic chemists?”, Angewandte Chemie International Edition, 48: 1030-1069.
  29. Regis, Y. N. G., Spyrou, K., Rudolf, P., (2010). “A roadmap to high quality chemically prepared graphene”, Journal of Physics D, 43: 374015-374034.
  30. Compton, O. C., Nguyen, S. T., (2010). “Graphene oxide, highly reduced graphene oxide, and graphene: versatile building blocks for carbon-based materials”, Small, 6: 711-723.
  31. Park, S., Ruoff, R. S., (2009). “Chemical methods for the production of graphenes”, Nature Nanotechnology, 4: 217-224.
  32. Zhu, Y., Murali, S., Cai, W., Li, X., Suk J. W., Potts, J. R., Ruof, R. S., (2010). “Graphene and Graphene oxide: synthesis, properties and applications”, Advanced Materials, 22: 3906-3924.
  33. Huang, X., Yin, Z., Wu, S., Qi, X., He, Q., Zhang, Q., Yan, Q., Boey, F., Zhang, H., (2011). “Graphene based materials: synthesis, characterization, properties, and applications”, Small, 7: 1876-1902.
  34. Guo, S., Dong, S., (2011). “Graphene nanosheet: synthesis, molecular engineering, thin film, hybrids, and energy and analytical applications”, Chemical Society Review, 40: 26442672.
  35. Neto, A. H. C., Guinea, F., Peres, N. M. R., Novoselov, K. S., Geim, A. K., (2009). “The electronic properties of graphene”, Reviews of Modern Physics, 81: 109-162.
  36. Geim, A. K., MacDonald, A. H., (2007). “Graphene: exploring carbon flatland”, Physics Today, 60: 35-41.
  37. Katsnelson, M. I., Novoselov, K. S., (2007). “Graphene: new bridge between condensed matter physics and quantum electrodynamics”, Solid State Community,143: 3-13.
  38. Rao, C. N. R., Sood, A. K., Subrahmanyam, K. S., Govindaraj, A., (2009). “Graphene: the new two-dimensional nanomaterial”, Angewandte Chemie International Edition, 48: 7752-7777.
  39. Loh, K. P., Bao, Q., Ang, P. K., Yang, J., (2010). “The chemistry of graphene”, Journal of Materials Chemistry, 20: 2277-2289.
  40. Boukhvalov, D. W., Katsnelson, M. I., (2009). “Chemical functionalization of graphene”, Journal of Phyics: Condensed Matter, 21: 344205-344217.
  41. Allen, M. J., Tung, V. C., Kaner, R. B., (2009). “Honeycomb carbon: a review of graphene”, Chemical Reviews, 110: 132-145.
  42. Huang, X., Qi, X., Boey, F., Zhang, H., (2012). “Graphene-based composites”, Chemical Society Reviews, 41: 666-686.
  43. Hummers, W. S., Offeman, R. E., (1958). “Preparation of graphitic oxide”, Journal of American Chemical Society, 80: 1339-1339.
  44. Farazas, A., Mavropoulos, A., Christofilos, D., Tsiaoussis, I., Tsipas, D., (2018). “Ultrasound Assisted Green Synthesis and Characterization of Graphene Oxide”, International Journal of Nanoscience and Nanotechnology, 14: 11-17.
  45. Fan, X., Peng, W., L,i Y., Li, X., Wang, S., Zhang, G., Zhang, F., (2008). “Deoxygenation of exfoliated graphite oxide under alkaline conditions: A green route to graphene preparation”, Advanced Materials, 20: 4490-4493.
  46. Stankovich, S., Dikin, D. A., Piner, R. D., Kohlhaas, K. A., Kleinhammes, A., Jia, Y., Wu, Y., Nguyen, S. T., Ruoff, R. S., (2007). “Synthesis of graphene-based nanosheets via chemical reduction of exfoliated graphite oxide”, Carbon, 45: 15581565.
  47. Zhu, C., Guo, S., Fang, Y., Dong, S., (2010). “Reducing sugar: New functional molecules for the green synthesis of graphene nanosheets”, ACS Nano, 4: 2429-2437.
  48. Zhang, J. L, Yang, H. J., Shen, G. X., Cheng, P., Zhang, J. Y., Guo S. W., (2010). “Reduction of graphene oxide via L-ascorbic acid”, Chemical Communnications, 46: 1112-1114.
  49. Wang, Z., Zhou, X., Zhang J., Boey, F., Zhang, H., (2009). “Direct electrochemical reduction of single-layer graphene oxide and subsequent functionalization with glucose oxidase”, The Journal of Physical Chemistry C, 113: 14071-14075.
  50. Zhou, M., Wang, Y., Zhai, Y., Zhai, J., Ren, W., Wang, F., Dong, S., (2009). “Controlled synthesis of large-area and patterned electrochemically reduced graphene oxide films”, Chemistry- A European Journal, 15: 6116-6120.
  51. Mcallister, M. J., Li, J., Adamson, H. D., Schnlepp, C. H., Abdalam, A. A., Liu, J., Herrera-Alonso, M., Milius, D. L., Car, R., Prud’gomme, R. K., Aksay, I. A., (2007). “Single sheet functionalized graphene by oxidation and thermal expansion of graphite”, Chemistry of Materials, 19: 4396-4404.
  52. Gao, J., Liu, F., Liu, Y., Ma, N., Wang, Z., Zhang, X., (2010). “Environment-friendly method to produce graphene that employs vitamin C and amino acid”, Chemistry of Materials, 22: 2213-2218.
  53. Guo, H., Wang, X. F., Qian, Q. Y., Wang, F. B., Xia, X. H., (2009). “A green approach to the synthesis of graphene nanosheets”, ACS Nano, 3: 2653-2659.
  54. Salas, E. C., Sun, Z., Luttge, A., Tour, J. M., (2010). “Reduction of graphene oxide via bacterial respiration”, ACS Nano, 4: 4852-4856.
  55. Wang, G., Qian, F., Saltikov, C., Jiao, Y., Li, Y., (2011). “Microbial reduction of graphene oxide by Shewanella”, Nano Research, 4: 563-570.
  56. Akhavan, O., Ghaderi, E., (2012). “Escherichia coli bacteria reduce graphene oxide to bactericidal graphene in a self-limiting manner”, Carbon, 50: 1853-1860.
  57. Gurunathan, S., Han, J. W., Eppakayala, V., Kim, J. H., (2013). “Microbial reduction of graphene oxide by Escherichia coli: a green chemistry approach”,  Colloids and Surfaces B: Biointerfaces, 102:772-777.
  58. Al Ali Alamaadeed, M., (2014). “Simple production method for graphene by microorganisms”, Patent No. US20150336799 A1.
  59. Khanra, P., Kuila, T., Kim, N. H., Bae, S. H., Yu, D. S., Lee, J. H., (2012). “Simultaneous biofunctionalization and reduction of graphene oxide by baker’s yeast”, Chemical Engineering Journal, 183: 526-570.
  60. Tucureanu, V., Matei, A., Avram, A. M., (2016). “FTIR Spectroscopy for Carbon Family Study”, Critical Reviews in Analytical Chemistry, 46: 502-520.
  61. Niyogi, S., Bekyarova, E., Itkis, M. E., Zhang, H., Shepperd, K., Hicks, J., Sprinkle, M., Berger, C., Lau, C. N., De Heer W. A., Conrad, E. H., Haddon, R. C., (2010). “Spectroscopy of covalently functionalized graphene”, Nano Letters, 10: 4061-4066.
  62. Frank, O., Mohr, M., Maultzsch, J., Thomsen, C., Riaz, I., Jalil, R., Novoselov, K. S., Tsoukleri, G., Parthenios, J., Papagelis, K., Kavan, L., Galiotis, C., (2011). “Raman 2D-band splitting in graphene: theory and experiment”, ACS Nano, 5: 2231-2239.
  63. Zhou, Y., Bao, Q., Tang, L. A. L., Zhong, Y., Loh, K. P., (2009). “Hydrothermal dehydration for the "green" reduction of exfoliated graphene oxide to graphene and demonstration of tunable optical limiting properties”, Chemistry of Materials, 21: 2950-2956.
  64. Brooijmans, R. J. W., De Vos, W. M., Hugenholtz, J., (2009). “Lactobacillus plantarum WCFS1 Electron Transport Chains”, Applied and Environmental Microbiology, 75: 3580-3585