A Novel Method to Reveal a Ureolytic Biofilm Attachment and In Situ Growth Monitoring by Electrochemical Impedance Spectroscopy


The formation of biofilms capable of efficiently carrying out ureolysis is of fundamental importance in several biotechnological systems such as urinary tract infections, building materials and municipal wastewater treatment. This work proposes a straightforward method for the formation of a ureolytic biofilm attached to graphite. The proposed strategy reduced the time needed to complete ureolysis to 3 days instead of 16 days required in suspension culture. To confirm the formation of a ureolytic biofilm, scanning electron microscopy and confocal laser scanning microscopy studies were employed ex situ. However, it is imperative to analyse the biofilm by direct non-invasive techniques. Accordingly, open circuit potential (OCP) and electrochemical impedance spectroscopy (EIS) were used as in situ monitoring techniques. The reduction in OCP from − 0.01 to − 0.2 V vs. Ag/AgCl and the increase in capacitance from 200 to 260 μF cm−2 were related to biofilm attachment. To the best of our knowledge, this is the first time in which a ureolytic biofilm attachment has been analysed by EIS. The increase in the biomass from 0.04 to 2.81 μm3 μm−2 and in average thickness from 10.19 to 32.78 μm was related to biofilm maturation.

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  1. 1.

    Phillips, A. J., Gerlach, R., Lauchnor, E., Mitchell, A. C., Cunningham, A. B., & Spangler, L. (2013). Engineered applications of ureolytic biomineralization: a review. Biofouling, 29(6), 715–733. https://doi.org/10.1080/08927014.2013.796550.

    CAS  Article  PubMed  Google Scholar 

  2. 2.

    Connolly, J. M., Jackson, B., Rothman, A. P., Klapper, I., & Gerlach, R. (2015). Estimation of a biofilm-specific reaction rate: kinetics of bacterial urea hydrolysis in a biofilm. Npj Biofilms and Microbiomes, 1 (1), 15014. https://doi.org/10.1038/npjbiofilms.2015.14.

  3. 3.

    Cuthbert, M. O., Riley, M. S., Handley-Sidhu, S., Renshaw, J. C., Tobler, D. J., Phoenix, V. R., & Mackay, R. (2012). Controls on the rate of ureolysis and the morphology of carbonate precipitated by S. Pasteurii biofilms and limits due to bacterial encapsulation. Ecological Engineering, 41, 32–40. https://doi.org/10.1016/j.ecoleng.2012.01.008.

    Article  Google Scholar 

  4. 4.

    Luther, A. K., Desloover, J., Fennell, D. E., & Rabaey, K. (2015). Electrochemically driven extraction and recovery of ammonia from human urine. Water Research, 87, 367–377. https://doi.org/10.1016/j.watres.2015.09.041.

    CAS  Article  PubMed  Google Scholar 

  5. 5.

    Mooshammer, M., Wanek, W., Hämmerle, I., Fuchslueger, L., Hofhansl, F., Knoltsch, A., Schnecker, J., Takriti, M., Watzka, M., Wild, B., Keiblinger, K. M., Zechmeister-Boltenstern, S., & Richter, A. (2014). Adjustment of microbial nitrogen use efficiency to carbon:nitrogen imbalances regulates soil nitrogen cycling. Nature Communications, 5(1), 1–7. https://doi.org/10.1038/ncomms4694.

    CAS  Article  Google Scholar 

  6. 6.

    Li, X., Lu, N., Brady, H. R., & Packman, A. I. (2016). Ureolytic biomineralization reduces Proteus mirabilis biofilm susceptibility to ciprofloxacin. Antimicrobial Agents and Chemotherapy, 60(5), 2993–3000. https://doi.org/10.1128/AAC.00203-16.

  7. 7.

    Jiang, N. J., Yoshioka, H., Yamamoto, K., & Soga, K. (2016). Ureolytic activities of a urease-producing bacterium and purified urease enzyme in the anoxic condition: implication for subseafloor sand production control by microbially induced carbonate precipitation (MICP). Ecological Engineering, 90, 96–104. https://doi.org/10.1016/j.ecoleng.2016.01.073.

  8. 8.

    Kanematsu, H., & Barry, D. M. (2015). Biofilm and Materials Science. (H. Kanematsu & D. M. Barry, Eds.) Biofilm and Materials Science. Cham: Springer International Publishing. https://doi.org/10.1007/978-3-319-14565-5.

  9. 9.

    Garrett, T. R., Bhakoo, M., & Zhang, Z. (2008). Bacterial adhesion and biofilms on surfaces. Progress in Natural Science, 18(9), 1049–1056. https://doi.org/10.1016/j.pnsc.2008.04.001.

    CAS  Article  Google Scholar 

  10. 10.

    Grohmann, E., & Vaishampayan, A. (2017). Techniques in studying biofilms and their characterization: microscopy to advanced imaging system in vitro and in situ. In Biofilms in Plant and Soil Health (pp. 215–230). Wiley-Blackwell. https://doi.org/10.1002/9781119246329.ch12.

  11. 11.

    Mizan, M. F. R., Bang, H.-J., Sadekuzzaman, M., Lee, N., Kim, T.-J., & Ha, S.-D. (2017). Molecular characteristics, biofilm-forming abilities, and quorum sensing molecules in Vibrio parahaemolyticus strains isolated from marine and clinical environments in Korea. Biofouling, 33(5), 369–378. https://doi.org/10.1080/08927014.2017.1316840.

    CAS  Article  PubMed  Google Scholar 

  12. 12.

    Neu, T. R., Manz, B., Volke, F., Dynes, J. J., Hitchcock, A. P., & Lawrence, J. R. (2010). Advanced imaging techniques for assessment of structure, composition and function in biofilm systems. FEMS Microbiology Ecology, 72(1), 1–21. https://doi.org/10.1111/j.1574-6941.2010.00837.x.

    CAS  Article  PubMed  Google Scholar 

  13. 13.

    Castro, L., Zhang, R., Muñoz, J. A., González, F., Blázquez, M. L., Sand, W., & Ballester, A. (2014). Characterization of exopolymeric substances (EPS) produced by Aeromonas hydrophila under reducing conditions. Biofouling., 30(4), 501–511. https://doi.org/10.1080/08927014.2014.892586.

    CAS  Article  PubMed  Google Scholar 

  14. 14.

    Schlafer, S., & Meyer, R. L. (2017). Confocal microscopy imaging of the biofilm matrix. Journal of Microbiological Methods, 138, 50–59. https://doi.org/10.1016/j.mimet.2016.03.002.

    Article  PubMed  Google Scholar 

  15. 15.

    Karimi, A., Karig, D., Kumar, A., & Ardekani, A. M. (2015). Interplay of physical mechanisms and biofilm processes: review of microfluidic methods. Lab on a Chip, 15(1), 23–42. https://doi.org/10.1039/C4LC01095G.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  16. 16.

    Nolte, K. A., Schwarze, J., & Rosenhahn, A. (2017). Microfluidic accumulation assay probes attachment of biofilm forming diatom cells. Biofouling, 33(7), 531–543. https://doi.org/10.1080/08927014.2017.1328058.

    CAS  Article  PubMed  Google Scholar 

  17. 17.

    Wolf, G., Crespo, J. G., & Reis, M. A. M. (2002). Optical and spectroscopic methods for biofilm examination and monitoring. Reviews in Environmental Science and Biotechnology, 1(3), 227–251. https://doi.org/10.1023/A:1021238630092.

    Article  Google Scholar 

  18. 18.

    Dheilly, A., Linossier, I., Darchen, A., Hadjiev, D., Corbel, C., & Alonso, V. (2008). Monitoring of microbial adhesion and biofilm growth using electrochemical impedancemetry. Applied Microbiology and Biotechnology, 79(1), 157–164. https://doi.org/10.1007/s00253-008-1404-7.

    CAS  Article  PubMed  Google Scholar 

  19. 19.

    Govindasamy, M., Wang, S. F., Subramanian, B., Ramalingam, R. J., Al-lohedan, H., & Sathiyan, A. (2019). A novel electrochemical sensor for determination of DNA damage biomarker (8-hydroxy-2′-deoxyguanosine) in urine using sonochemically derived graphene oxide sheets covered zinc oxide flower modified electrode. Ultrasonics Sonochemistry, 58. https://doi.org/10.1016/j.ultsonch.2019.104622.

  20. 20.

    Govindasamy, M., Wang, S. F., Kumaravel, S., Ramalingam, R. J., & Al-lohedan, H. A. (2019). Facile synthesis of copper sulfide decorated reduced graphene oxide nanocomposite for high sensitive detection of toxic antibiotic in milk. Ultrasonics Sonochemistry, 52, 382–390. https://doi.org/10.1016/j.ultsonch.2018.12.015.

    CAS  Article  PubMed  Google Scholar 

  21. 21.

    Vinoth, S., Govindasamy, M., Wang, S. F., & Anandaraj, S. (2020). Layered nanocomposite of zinc sulfide covered reduced graphene oxide and their implications for electrocatalytic applications. Ultrasonics Sonochemistry, 64. https://doi.org/10.1016/j.ultsonch.2020.105036.

  22. 22.

    Anderson, K., Sallis, P., & Uyanik, S. (2003). Anaerobic treatment processes. In N. H. Duncan Mara (Ed.), Handbook of Water and Wastewater Microbiology (pp. 391–426). Amsterdam: Elsevier. https://doi.org/10.1016/B978-012470100-7/50025-X.

    Chapter  Google Scholar 

  23. 23.

    Madhuri, R. J., Saraswathi, M., Gowthami, K., Bhargavi, M., Divya, Y., & Deepika, V. (2019). Recent approaches in the production of novel enzymes from environmental samples by enrichment culture and metagenomic approach. In V. Buddolla (Ed.), Recent Developments in Applied Microbiology and Biochemistry (pp. 251–262). Elsevier, Amsterdam. https://doi.org/10.1016/B978-0-12-816328-3.00019-2.

  24. 24.

    Yan, L., & Xing, W. (2018). Methods to study magnetotactic bacteria and magnetosomes. In J. T. T. Volker Gurtler (Ed.), Methods in Microbiology (pp. 357–386). https://doi.org/10.1016/bs.mim.2018.05.003.

  25. 25.

    Mobley, H. L., & Hausinger, R. P. (1989). Microbial ureases: significance, regulation, and molecular characterization. Microbiological Reviews, 53(1), 85–108.

    CAS  Article  Google Scholar 

  26. 26.

    Madigan, M. T., Martinko, J. M., Bender, K. S., Buckley, D. H., & Stahl, D. A. (2015). Brock Biology of Microorganisms. (Pearson, Ed.) (Fourteenth.). Pearson. Retrieved from https://books.google.com.mx/books?id=pAhVnwEACAAJ

  27. 27.

    Stuart, C. A., Van Stratum, E., & Rustigian, R. (1945). Further studies on urease production by Proteus and related organisms. Journal of Bacteriology, 49(5), 437–444 Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/16560936.

    CAS  Article  Google Scholar 

  28. 28.

    Kuntke, P., Śmiech, K. M., Bruning, H., Zeeman, G., Saakes, M., Sleutels, T. H. J. A., Hamelers, H. V. M., & Buisman, C. J. N. (2012). Ammonium recovery and energy production from urine by a microbial fuel cell. Water Research, 46(8), 2627–2636. https://doi.org/10.1016/j.watres.2012.02.025.

    CAS  Article  PubMed  Google Scholar 

  29. 29.

    Hussain Qadri, S. M., Zubairi, S., Hawley, H. P., Mazlaghani, H. H., & Ramirez, E. G. (1984). Rapid test for determination of urea hydrolysis. Antonie Van Leeuwenhoek, 50(4), 417–423. https://doi.org/10.1007/BF00394656.

    CAS  Article  Google Scholar 

  30. 30.

    Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72(1–2), 248–254.

    CAS  Article  Google Scholar 

  31. 31.

    Matsukawa, M., & Greenberg, E. P. (2004). Putative exopolysaccharide synthesis genes influence Pseudomonas aeruginosa biofilm development. Journal of Bacteriology, 186(14), 4449–4456. https://doi.org/10.1128/JB.186.14.4449-4456.2004.

  32. 32.

    Schönleber, M., Klotz, D., & Ivers-Tiffée, E. (2014). A method for improving the robustness of linear Kramers-Kronig validity tests. Electrochimica Acta, 131, 20–27. https://doi.org/10.1016/j.electacta.2014.01.034.

  33. 33.

    Boukamp, B. A. (1995). A linear Kronig-Kramers transform test for immittance data validation. Journal of the Electrochemical Society, 142(6), 1885. https://doi.org/10.1149/1.2044210.

  34. 34.

    Méndez-Tovar, M., García-Meza, J. V., & González, I. (2019). Electrochemical monitoring of Acidithiobacillus thiooxidans biofilm formation on graphite surface with elemental sulfur. Bioelectrochemistry, 128, 30–38. https://doi.org/10.1016/j.bioelechem.2019.03.004.

  35. 35.

    Heydorn, A., Nielsen, A. T., Hentzer, M., Sternberg, C., Givskov, M., Ersbøll, B. K., & Molin, S. (2000). Quantification of biofilm structures by the novel computer program COMSTAT. Microbiology, 146(10), 2395–2407.

    CAS  Article  Google Scholar 

  36. 36.

    Vorregaard, M. (2008). Comstat2 - a modern 3D image analysis environment for biofilms. PhD thesis.

  37. 37.

    Geisseler, D., Horwath, W. R., Joergensen, R. G., & Ludwig, B. (2010). Pathways of nitrogen utilization by soil microorganisms - a review. Soil Biology and Biochemistry, 42(12), 2058–2067. https://doi.org/10.1016/j.soilbio.2010.08.021.

    CAS  Article  Google Scholar 

  38. 38.

    Pérez, E. J., Cabrera-Sierra, R., González, I., & Ramírez-Vives, F. (2007). Influence of Desulfovibrio sp. biofilm on SAE 1018 carbon steel corrosion in synthetic marine medium. Corrosion Science, 49(9), 3580–3597. https://doi.org/10.1016/j.corsci.2007.03.034.

    CAS  Article  Google Scholar 

  39. 39.

    Zamora, P., Georgieva, T., Ter Heijne, A., Sleutels, T. H. J. A., Jeremiasse, A. W., Saakes, M., … Kuntke, P. (2017). Ammonia recovery from urine in a scaled-up microbial electrolysis cell. Journal of Power Sources, 356, 491–499. https://doi.org/10.1016/j.jpowsour.2017.02.089.

  40. 40.

    Anthonisen, A. C., Loehr, R. C., Prakasam, T. B. S., & Srinath, E. G. (1976). Inhibition of nitrification by ammonia and nitrous acid. Source Journal (Water Pollution Control Federation), 48(5), 835–852. https://doi.org/10.1017/CBO9781107415324.004.

    CAS  Article  Google Scholar 

  41. 41.

    Rodríguez Arredondo, M., Kuntke, P., ter Heijne, A., Hamelers, H. V. M., & Buisman, C. J. N. (2017). Load ratio determines the ammonia recovery and energy input of an electrochemical system. Water Research, 111, 330–337. https://doi.org/10.1016/j.watres.2016.12.051.

    CAS  Article  PubMed  Google Scholar 

  42. 42.

    Kim, T., Kang, J., Lee, J.-H., & Yoon, J. (2011). Influence of attached bacteria and biofilm on double-layer capacitance during biofilm monitoring by electrochemical impedance spectroscopy. Water Research, 45(15), 4615–4622. https://doi.org/10.1016/j.watres.2011.06.010.

    CAS  Article  PubMed  Google Scholar 

  43. 43.

    Babauta, J. T., & Beyenal, H. (2014). Mass transfer studies of Geobacter sulfurreducens biofilms on rotating disk electrodes. Biotechnology and Bioengineering, 111(2), 285–294. https://doi.org/10.1002/bit.25105.

    CAS  Article  PubMed  Google Scholar 

  44. 44.

    Lvovich, V. F. (2012). Fundamentals of electrochemical impedance spectroscopy. Impedance spectroscopy (pp. 1–21). New York: Wiley.

    Google Scholar 

  45. 45.

    Babauta, J. T., & Beyenal, H. (2017). Use of a small overpotential approximation to analyze Geobacter sulfurreducens biofilm impedance. Journal of Power Sources, 356, 549–555. https://doi.org/10.1016/j.jpowsour.2017.03.021.

    CAS  Article  Google Scholar 

  46. 46.

    Manohar, A. K., Bretschger, O., Nealson, K. H., & Mansfeld, F. (2008). The use of electrochemical impedance spectroscopy (EIS) in the evaluation of the electrochemical properties of a microbial fuel cell. Bioelectrochemistry, 72(2), 149–154. https://doi.org/10.1016/j.bioelechem.2008.01.004.

    CAS  Article  PubMed  Google Scholar 

  47. 47.

    Naumowicz, M., Petelska, A. D., & Figaszewski, Z. A. (2011). Impedance analysis of complex formation equilibria in phosphatidylcholine bilayers containing decanoic acid or decylamine. Cell Biochemistry and Biophysics, 61(1), 145–155. https://doi.org/10.1007/s12013-011-9171-y.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  48. 48.

    Cui, X., & Martin, D. C. (2003). Electrochemical deposition and characterization of poly(3,4-ethylenedioxythiophene) on neural microelectrode arrays. Sensors and Actuators, B: Chemical, 89(1–2), 92–102. https://doi.org/10.1016/S0925-4005(02)00448-3.

    CAS  Article  Google Scholar 

  49. 49.

    ter Heijne, A., Schaetzle, O., Gimenez, S., Navarro, L., Hamelers, B., & Fabregat-Santiago, F. (2015). Analysis of bio-anode performance through electrochemical impedance spectroscopy. Bioelectrochemistry, 106(Pt A), 64–72. https://doi.org/10.1016/j.bioelechem.2015.04.002.

    CAS  Article  PubMed  Google Scholar 

  50. 50.

    Maurício, R., Dias, C. J., & Santana, F. (2006). Monitoring biofilm thickness using a non-destructive, on-line, electrical capacitance technique. Environmental Monitoring and Assessment, 119(1–3), 599–607. https://doi.org/10.1007/s10661-005-9045-0.

    CAS  Article  PubMed  Google Scholar 

  51. 51.

    Bimakr, F., Ginige, M. P., Kaksonen, A. H., Sutton, D. C., Puzon, G. J., & Cheng, K. Y. (2018). Assessing graphite and stainless-steel for electrochemical sensing of biofilm growth in chlorinated drinking water systems. Sensors and Actuators, B: Chemical, 277, 526–534. https://doi.org/10.1016/j.snb.2018.09.005.

    CAS  Article  Google Scholar 

  52. 52.

    Huerta-Miranda, G. A., Arroyo-Escoto, A. I., Burgos, X., Juárez, K., & Miranda-Hernández, M. (2019). Influence of the major pilA transcriptional regulator in electrochemical responses of Geobacter sulfureducens PilR-deficient mutant biofilm formed on FTO electrodes. Bioelectrochemistry, 127, 145–153. https://doi.org/10.1016/j.bioelechem.2019.02.006.

  53. 53.

    Zhou, Y., & Gao, X. (2019). Characterization of biofilm formed by phenanthrene-degrading bacteria on rice root surfaces for reduction of pah contamination in rice. International Journal of Environmental Research and Public Health, 16(11). https://doi.org/10.3390/ijerph16112002.

  54. 54.

    Horemans, B., Hofkens, J., Smolders, E., & Springael, D. (2014). Biofilm formation of a bacterial consortium on linuron at micropollutant concentrations in continuous flow chambers and the impact of dissolved organic matter. FEMS Microbiology Ecology, 88(1), 184–194. https://doi.org/10.1111/1574-6941.12280.

    CAS  Article  PubMed  Google Scholar 

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The authors would like to thank David Sepúlveda-Sánchez from the Laboratory of Electron Microscopy (UAM-I). They also acknowledge María de Jesús Perea Flores from the Multidisciplinary Laboratory of Characterisation of Nanostructures and Materials (IPN) for her support in confocal laser scanning microscopy. Additionally, the authors would like to thank Guillermo Huerta-Miranda from IBT-IER-UNAM for confocal image analysis.


This research was supported by the National Council for Science and Technology (CONACyT), Mexico, and scholarship 328072.

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Correspondence to Florina Ramírez.

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Romero, M.C., Ramos, G., González, I. et al. A Novel Method to Reveal a Ureolytic Biofilm Attachment and In Situ Growth Monitoring by Electrochemical Impedance Spectroscopy. Appl Biochem Biotechnol 193, 1379–1396 (2021). https://doi.org/10.1007/s12010-020-03386-8

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  • Ureolytic biofilm
  • Ureolysis
  • Ammonium recovery
  • Enrichment culture
  • Bacterial attachment
  • Electrochemical impedance spectroscopy