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An improvised microtiter dish biofilm assay for non-invasive biofilm detection on microbial fuel cell anodes and studying biofilm growth conditions

  • Environmental Microbiology - Research Paper
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Abstract

Microbial life is predominantly observed as biofilms, which are a sessile aggregation of microbial cells formed in response to stress conditions. The microtiter dish biofilm formation assay is one of the most important methods of studying biofilm formation. In this study, the assay has been improvised to allow easy detection of biofilm formation on different substrata. The method has then been used to study growth conditions that affect biofilm formation, viz., the effect of pH, temperature, shaking conditions, and the carbon source provided. Glass, cellulose acetate, and carbon cloth materials were used as substrata to study biofilm development under the above conditions. The method was then extended to determine biofilm formation on the anodes of a microbial fuel cell in order to study the effect of biofilm formation on power production. A high correlation was observed between biofilm formation and power density (r = 0.951). When the electrode containing a biofilm was replaced with another electrode without biofilm, the average power density dropped from 59.55 to 5.76 mW/m2. This method offers an easy way to study the suitability of different materials to support biofilm formation. Growth conditions determining biofilm formation can be studied using this method. This method also offers a non-invasive way to determine biofilm formation on anodes of microbial fuel cells and preserves the anode for further studies.

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References

  1. Merritt JH, Kadouri DE, O’Toole GA (2011) Growing and analyzing static biofilms. Curr Protoc Microbiol 22(SUPPL 22):1–18. https://doi.org/10.1002/9780471729259.mc01b01s22

    Article  Google Scholar 

  2. Flemming H-C, Wingender J, Szewzyk U, Steinberg P, Rice SA, Kjelleberg S (2016) Biofilms: an emergent form of bacterial life. Nat Rev Microbiol 14(9):563–575. https://doi.org/10.1038/nrmicro.2016.94

    Article  CAS  PubMed  Google Scholar 

  3. Jefferson KK (2004) What drives bacteria to produce a biofilm? FEMS Microbiol Lett 236(2):163–173. https://doi.org/10.1016/j.femsle.2004.06.005

    Article  CAS  PubMed  Google Scholar 

  4. Kumar R, Singh L, Wahid ZA, Din MFM (2015) Exoelectrogens in microbial fuel cells toward bioelectricity generation: a review. Int J Energy Res 39(8):1048–1067. https://doi.org/10.1002/er.3305

    Article  CAS  Google Scholar 

  5. Sutherland IW (2001) The biofilm matrix - an immobilized but dynamic microbial environment. Trends Microbiol 9(5):222–227. https://doi.org/10.1016/S0966-842X(01)02012-1

    Article  CAS  PubMed  Google Scholar 

  6. Wilkins M, Hall-Stoodley L, Allan RN, Faust SN (2014) New approaches to the treatment of biofilm-related infections. J Infect 69(S1):S47–S52. https://doi.org/10.1016/j.jinf.2014.07.014

    Article  PubMed  Google Scholar 

  7. Percival SL, Suleman L, Donelli G (2015) Healthcare-associated infections, medical devices and biofilms: risk, tolerance and control. J Med Microbiol 64(4):323–334. https://doi.org/10.1099/jmm.0.000032

    Article  PubMed  Google Scholar 

  8. Aiyer KS, Vijayakumar BS, Vishwanathan AS (2018) The enigma of biofilms. Curr Sci 115(2):204–205

    Article  CAS  Google Scholar 

  9. Falkowski PG, Fenchel T, Delong EF (2008) The microbial engines that drive Earth’s biogeochemical cycles. Science 320(5879):1034–1039. https://doi.org/10.1126/science.1153213

    Article  CAS  PubMed  Google Scholar 

  10. Godia F, Sola C (1995) Fluidized-bed bioreactors. Biotechnol Prog 11(5):479–497. https://doi.org/10.1021/bp00035a001

    Article  CAS  Google Scholar 

  11. Logan BE, Hamelers B, Rozendal R, Schröder U, Keller J, Freguia S, Aelterman P, Verstraete W, Rabaey K (2006) Microbial fuel cells: methodology and technology †. Environ Sci Technol 40(17):5181–5192. https://doi.org/10.1021/es0605016

    Article  CAS  PubMed  Google Scholar 

  12. Gude VG (2016) Wastewater treatment in microbial fuel cells – an overview. J Clean Prod 122:287–307. https://doi.org/10.1016/j.jclepro.2016.02.022

    Article  CAS  Google Scholar 

  13. Azeredo J, Azevedo NF, Briandet R, Cerca N, Coenye T, Costa AR, Desvaux M, di Bonaventura G, Hébraud M, Jaglic Z, Kačániová M, Knøchel S, Lourenço A, Mergulhão F, Meyer RL, Nychas G, Simões M, Tresse O, Sternberg C (2017) Critical review on biofilm methods. Crit Rev Microbiol 43(3):313–351. https://doi.org/10.1080/1040841X.2016.1208146

    Article  CAS  PubMed  Google Scholar 

  14. Pettit RK, Weber CA, Kean MJ, Hoffmann H, Pettit GR, Tan R, Franks KS, Horton ML (2005) Microplate Alamar blue assay for Staphylococcus epidermidis biofilm susceptibility testing. Antimicrob Agents Chemother 49(7):2612–2617. https://doi.org/10.1128/AAC.49.7.2612-2617.2005

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Donovan C, Dewan A, Heo D, Beyenal H (2008) Batteryless, wireless sensor powered by a sediment microbial fuel cell. Environ Sci Technol 42(22):8591–8596. https://doi.org/10.1021/es801763g

    Article  CAS  PubMed  Google Scholar 

  16. Feoktistova M, Geserick P, Leverkus M (2016) Crystal violet assay for determining viability of cultured cells. Cold Spring Harb Protoc 2016(4):343–346. https://doi.org/10.1101/pdb.prot087379

    Article  Google Scholar 

  17. Beecroft NJ, Zhao F, Varcoe JR, Slade RCT, Thumser AE, Avignone-Rossa C (2012) Dynamic changes in the microbial community composition in microbial fuel cells fed with sucrose. Appl Microbiol Biotechnol 93(1):423–437. https://doi.org/10.1007/s00253-011-3590-y

    Article  CAS  PubMed  Google Scholar 

  18. O’Toole GA (2011) Microtiter dish biofilm formation assay. J Vis Exp (47):3–5. https://doi.org/10.3791/2437

  19. Bower CK, Mcguire J, Daeschel MA (1996) The adhesion and detachment of bacteria and spores on food-contact surfaces. Trends Food Sci Technol 7(5):152–157. https://doi.org/10.1016/0924-224481255-6

    Article  CAS  Google Scholar 

  20. Lobelle D, Cunliffe M (2011) Early microbial biofilm formation on marine plastic debris. Mar Pollut Bull 62(1):197–200. https://doi.org/10.1016/j.marpolbul.2010.10.013

    Article  CAS  PubMed  Google Scholar 

  21. Burton E, Yakandawala N, LoVetri K, Madhyastha MS (2007) A microplate spectrofluorometric assay for bacterial biofilms. J Ind Microbiol Biotechnol 34(1):1–4. https://doi.org/10.1007/s10295-006-0086-3

    Article  CAS  PubMed  Google Scholar 

  22. Billings N, Birjiniuk A, Samad TS, Doyle PS, Ribbeck K (2015) Material properties of biofilms—a review of methods for understanding permeability and mechanics. Rep Prog Phys 78(3):036601. https://doi.org/10.1088/0034-4885/78/3/036601

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Stoodley P, DeBeer D, Lappin - Scott H (1997) Influence of electric fields and pH on biofilm structure as related to the bioelectric effect. Antimicrob Agents Chemother 41(9):1876–1879

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Harvey J, Keenan KP, Gilmour A (2007) Assessing biofilm formation by listeria monocytogenes strains. Food Microbiol 24(4):380–392. https://doi.org/10.1016/j.fm.2006.06.006

    Article  CAS  PubMed  Google Scholar 

  25. Moreira JMR, Gomes LC, Araújo JDP, Miranda JM, Simões M, Melo LF, Mergulhão FJ (2013) The effect of glucose concentration and shaking conditions on Escherichia coli biofilm formation in microtiter plates. Chem Eng Sci 94:192–199. https://doi.org/10.1016/j.ces.2013.02.045

    Article  CAS  Google Scholar 

  26. Gristina AG, Costerton JW (1985) Bacterial adherence to biomaterials and tissue. The significance of its role in clinical sepsis. J Bone Joint Surg 67(2):264–273. https://doi.org/10.2106/00004623-198567020-00014

    Article  CAS  PubMed  Google Scholar 

  27. Villaverde S, García-Encina PA, Fdz-Polanco F (1997) Influence of pH over nitrifying biofilm activity in submerged biofilters. Water Res 31(5):1180–1186. https://doi.org/10.1016/S0043-1354(96)00376-4

    Article  CAS  Google Scholar 

  28. Römling U, Kjelleberg S, Normark S, Nyman L, Uhlin BE, Åkerlund B (2014) Microbial biofilm formation: a need to act. J Intern Med 276(2):98–110. https://doi.org/10.1111/joim.12242

    Article  PubMed  Google Scholar 

  29. Peyton BM (1996) Effects of shear stress and substrate loading rate on Pseudomonas aeruginosa biofilm thickness and density. Water Res 30(1):29–36. https://doi.org/10.1016/0043-1354(95)00110-7

    Article  CAS  Google Scholar 

  30. Stoodley P, Dodds I, Boyle JD, Lappin-Scott HM (1998) Influence of hydrodynamics and nutrients on biofilm structure. J Appl Microbiol 85(S1):19S–28S. https://doi.org/10.1111/j.1365-2672.1998.tb05279.x

    Article  PubMed  Google Scholar 

  31. Stepanović S, Vuković D, Dakić I, Savić B, Švabić-Vlahović M (2000) A modified microtiter-plate test for quantification of staphylococcal biofilm formation. J Microbiol Methods 40(2):175–179. https://doi.org/10.1016/S0167-7012(00)00122-6

    Article  PubMed  Google Scholar 

  32. Mohamed JA, Huang DB (2007) Biofilm formation by enterococci. J Med Microbiol 56(12):1581–1588. https://doi.org/10.1099/jmm.0.47331-0

    Article  CAS  PubMed  Google Scholar 

  33. van der Kooij D, Veenendaal HR, Baars-Lorist C, van der Klift DW, Drost YC (1995) Biofilm formation on surfaces of glass and Teflon exposed to treated water. Water Res 29(7):1655–1662. https://doi.org/10.1016/0043-1354(94)00333-3

    Article  Google Scholar 

  34. Rochex A, Lebeault J-M (2007) Effects of nutrients on biofilm formation and detachment of a Pseudomonas putida strain isolated from a paper machine. Water Res 41(13):2885–2892. https://doi.org/10.1016/j.watres.2007.03.041

    Article  CAS  PubMed  Google Scholar 

  35. Duetz WA, Witholt B (2004) Oxygen transfer by orbital shaking of square vessels and deepwell microtiter plates of various dimensions. Biochem Eng J 17(3):181–185. https://doi.org/10.1016/S1369-703X(03)00177-3

    Article  CAS  Google Scholar 

  36. Kumamoto CA (2002) Candida biofilms. Curr Opin Microbiol 5(6):608–611. https://doi.org/10.1016/S1369-5274(02)00371-5

    Article  CAS  PubMed  Google Scholar 

  37. Stepanović S, Ćirković I, Mijač V, Švabić-Vlahović M (2003) Influence of the incubation temperature, atmosphere and dynamic conditions on biofilm formation by Salmonella spp. Food Microbiol 20(3):339–343. https://doi.org/10.1016/S0740-0020(02)00123-5

    Article  Google Scholar 

  38. Kumar R, Singh L, Zularisam AW, Hai FI (2018) Microbial fuel cell is emerging as a versatile technology: a review on its possible applications, challenges and strategies to improve the performances. Int J Energy Res 42(2):369–394. https://doi.org/10.1002/er.3780

    Article  Google Scholar 

  39. Xiao Y, Zhao F (2017) Electrochemical roles of extracellular polymeric substances in biofilms. Curr Opin Electrochem 4(1):206–211. https://doi.org/10.1016/j.coelec.2017.09.016

    Article  CAS  Google Scholar 

  40. Patil SA, Hägerhäll C, Gorton L (2012) Electron transfer mechanisms between microorganisms and electrodes in bioelectrochemical systems. In: Advances in chemical bioanalysis, vol 1. Springer International Publishing, Cham, pp 71–129. https://doi.org/10.1007/11663_2013_2

    Chapter  Google Scholar 

  41. Schröder U (2007) Anodic electron transfer mechanisms in microbial fuel cells and their energy efficiency. Phys Chem Chem Phys 9(21):2619–2629. https://doi.org/10.1039/b703627m

    Article  CAS  PubMed  Google Scholar 

  42. Logan BE (2009) Exoelectrogenic bacteria that power microbial fuel cells. Nat Rev Microbiol 7(5):375–381. https://doi.org/10.1038/nrmicro2113

    Article  CAS  PubMed  Google Scholar 

  43. Saratale GD, Saratale RG, Shahid MK, Zhen G, Kumar G, Shin H-S, Choi YG, Kim S-H (2017) A comprehensive overview on electro-active biofilms, role of exo-electrogens and their microbial niches in microbial fuel cells (MFCs). Chemosphere 178:534–547. https://doi.org/10.1016/j.chemosphere.2017.03.066

    Article  CAS  PubMed  Google Scholar 

  44. Reguera G, Nevin KP, Nicoll JS, Covalla SF, Woodard TL, Lovley DR (2006) Biofilm and nanowire production leads to increased current in Geobacter sulfurreducens fuel cells. Appl Environ Microbiol 72(11):7345–7348. https://doi.org/10.1128/AEM.01444-06

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgments

The authors dedicate this work to Bhagawan Sri Sathya Sai Baba, the founder chancellor of Sri Sathya Sai Institute of Higher Learning.

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  1. Department of Biosciences, Sri Sathya Sai Institute of Higher Learning, Prasanthi Nilayam, Puttaparthi, Andhra Pradesh, 515134, India

    Kartik S. Aiyer & B. S. Vijayakumar

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  1. Kartik S. Aiyer
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Correspondence to Kartik S. Aiyer.

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Aiyer, K.S., Vijayakumar, B.S. An improvised microtiter dish biofilm assay for non-invasive biofilm detection on microbial fuel cell anodes and studying biofilm growth conditions. Braz J Microbiol 50, 769–775 (2019). https://doi.org/10.1007/s42770-019-00091-5

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