Applied Microbiology and Biotechnology

, Volume 100, Issue 6, pp 2855–2868 | Cite as

Assessment of microbial viability in municipal sludge following ultrasound and microwave pretreatments and resulting impacts on the efficiency of anaerobic sludge digestion

  • Monica Angela Cella
  • Deniz Akgul
  • Cigdem EskiciogluEmail author
Environmental biotechnology


A range of ultrasonication (US) and microwave irradiation (MW) sludge pretreatments were compared to determine the extent of cellular destruction in micro-organisms within secondary sludge and how this cellular destruction translated to anaerobic digestion (AD). Cellular lysis/inactivation was measured using two microbial viability assays, (1) Syto 16® Green and Sytox® Orange counter-assay to discern the integrity of cellular membranes and (2) a fluorescein diacetate assay to understand relative enzymatic activity. A range of MW intensities (2.17–6.48 kJ/g total solids or TS, coinciding temperatures of 60–160 °C) were selected for comparison via viability assays; a range of corresponding US intensities (2.37–27.71 kJ/g TS, coinciding sonication times of 10–60 min at different amplitudes) were also compared to this MW range. The MW pretreatment of thickened waste activated sludge (tWAS) caused fourfold to fivefold greater cell death than non-pretreated and US-pretreated tWAS. The greatest microbial destruction occurred at MW intensities greater than 2.62 kJ/g TS of sludge, after which increased energy input via MW did not appear to cause greater microbial death. In addition, the optimal MW pretreatment (80 °C, 2.62 kJ/g TS) and corresponding US pretreatment (10 min, 60 % amplitude, 2.37 kJ/g TS) were administered to the tWAS of a mixed sludge and fed to anaerobic digesters over sludge retention times (SRTs) of 20, 14, and 7 days to compare effects of feed pretreatment on AD efficiency. The digester utilizing MW-pretreated tWAS (80 °C, 2.62 kJ/g TS) had the greatest fecal coliform removal (73.4 and 69.8 % reduction, respectively), greatest solids removal (44.2 % TS reduction), and highest overall methane production (248.2 L CH4/kg volatile solids) at 14- and 7-day SRTs. However, despite the fourfold to fivefold increases in cell death upon pretreatment, improvements from the digester fed MW-pretreated sludge were marginal (i.e., increases in efficiency of less than 3–10 %) and likely due to a smaller proportion of cells (10–20 %) in the polymeric network and mixed sludge fed to digesters.


Anaerobic digestion Sludge pretreatment Microwave irradiation Ultrasonication Microbial viability Municipal waste 



The authors would like to thank the City of Kelowna and the Government of British Columbia Ministry of Environment for supporting this project. This work has been carried out under the sponsorships of the Natural Science and Engineering Council of Canada (NSERC) Strategic Project Grant (#396519-10) and the financial support of the Scientific and Technological Research Council of Turkey (TUBITAK).


  1. American Public Health Association (APHA) (2005) Standard methods for the examination of water and wastewater. Washington DC USA.Google Scholar
  2. Appels L, Baeyens J, Degrève J, Dewil R (2008) Principles and potential of the anaerobic digestion of waste-activated sludge. Prog Energy Combust Sci 34:755–781CrossRefGoogle Scholar
  3. Biggerstaff JP, Le Puil M, Weidow BL, Prater J, Glass K, Radosevich M, White DC (2006) New methodology for viability testing in environmental samples. Mol Cell Probes 20:141–146CrossRefPubMedGoogle Scholar
  4. Bougrier C, Carrère H, Delgenes JP (2005) Solubilisation of waste-activated sludge by ultrasonic treatment. Chem Eng J 106(2):163–169CrossRefGoogle Scholar
  5. British Columbia Organic Matter Recycling Regulation (BC OMRR) 2008 Land application guidelines for the organic matter recycling regulation and the soil amendment code of practice, best management practice. BC OMRR and Soil Amendment Code of Practice (SACoP) Land Application Guidelines. Prepared for: BC Ministry of Environment. Prepared by: SYLVIS Environmental. New Westminster, British Columbia.Google Scholar
  6. Cai Y, Strømme M, Welch K (2014) Bacteria viability assessment after photocatalytic treatment. Biotechnology. 3 Biotech 4:149–157Google Scholar
  7. Carrère H, Dumas C, Battimelli A, Batstone DJ, Delgenès JP, Steyer JP, Ferrer I (2010) Pretreatment methods to improve sludge anaerobic degradability: a review. J Hazard Mater 183:1–15CrossRefPubMedGoogle Scholar
  8. Chu CP, Lee DJ, Chang BV, You CS, Tay JH (2002) “Weak” ultrasonic pretreatment on anaerobic digestion of flocculated activated biosolids. Water Res 36(11):2681–2688CrossRefPubMedGoogle Scholar
  9. Dholiya K, Patel D, Kothari V (2012) Effect of low power microwave on microbial growth, enzyme activity, and aflatoxin production. Research in Biotechnology 3(4):28–34Google Scholar
  10. Dreyfuss MS, Chipley JR (1980) Comparison of effects of sublethal microwave irradiation and conventional heating on the metabolic activity of Staphylococcus aureus. Appl Environ Microbiol 39:13–16PubMedCentralPubMedGoogle Scholar
  11. Droste RL (1997) Theory and practice of water and wastewater treatment. John Wiley & Sons, New Jersey, USAGoogle Scholar
  12. Eskicioglu C, Terzian N, Kennedy KJ, Droste RL, Hamoda M (2007) Athermal microwave effects for enhancing digestibility of waste activated sludge. Water Res 41:2457–2466CrossRefPubMedGoogle Scholar
  13. Fleming, J. (1961) Microwave irradiation in relation to biological systems and neural activity. In: Biological effects of microwave radiation. Plenum Press, New York, USA.Google Scholar
  14. Hosseini Koupaie EH, Eskicioglu C (2015) Below and above boiling point comparison of microwave irradiation and conductive heating for municipal sludge digestion under identical heating/cooling profiles. Bioresour Technol 187:235–245CrossRefPubMedGoogle Scholar
  15. Houtmeyers, S., Appels, L., Degrève, J., Van Impe, J., Dewil, R. (2013) Comparing the influence of ultrasonic and microwave pretreatment on the solubilization and semi-continuous digestion of waste activated sludge. Proceedings of the anaerobic digestion conference, Santiago de Compostela, Spain.Google Scholar
  16. Lew S, Lew M, Mieszczyński T, Szarek J (2010) Selected fluorescent techniques for identification of the physiological state of individual water and soil bacterial cells—review. Folia Microbiol 55(2):107–118CrossRefGoogle Scholar
  17. Mata-Alvarez J, Macé S, Llabrés P (2000) Anaerobic digestion of organic solid wastes. An overview of research achievements and perspectives. Bioresour Technol 74:3–16CrossRefGoogle Scholar
  18. Mehdizadeh SN, Eskicioglu C, Bobowski J, Johnson T (2013) Conductive heating and microwave hydrolysis under identical heating profiles for advanced anaerobic digestion of municipal sludge. Water Res 47:5040–5051CrossRefPubMedGoogle Scholar
  19. Narihiro T, Sekiguchi Y (2007) Microbial communities in anaerobic digestion processes for waste and wastewater treatment: a microbiological update. Curr Opin Biotechnol 18(3):273–278CrossRefPubMedGoogle Scholar
  20. Nielson PH (2002) In: Bitton G (ed) Activated sludge—the floc. Encyclopedia of Environmental Microbiology. Wiley-Interscience, Hoboken, New Jersey, USAGoogle Scholar
  21. Pascaud A, Amellal S, Soulas ML, Soulas G (2009) A fluorescence-based assay for measuring the viable cell concentration of mixed microbial communities in soil. J Microbiol Methods 76:81–87CrossRefPubMedGoogle Scholar
  22. Peeters E, Nelis HJ, Coenye T (2008) Comparison of multiple methods for quantification of microbial biofilms grown in microtiter plates. J Microbiol Methods 72:157–165CrossRefPubMedGoogle Scholar
  23. Pino-Jelcic SA, Hong SM, Park JK (2006) Enhanced anaerobic biodegradability and inactivation of fecal coliforms and Salmonella spp. in wastewater sludge by using microwaves. Water Environment Research 78(2):209–216CrossRefPubMedGoogle Scholar
  24. Prorot A, Eskicioglu C, Droste R, Dagot C, Leprat P (2008) Assessment of physiological state of micro-organisms in activated sludge with flow cytometry: application for monitoring sludge production minimization. J Ind Microbiol Biotechnol 35:1261–1268CrossRefPubMedGoogle Scholar
  25. Rocher M, Goma G, Pilas Begue A, Louvel L, Rols JL (1999) Towards a reduction in excess sludge production in activated sludge processes: biomass physicochemical treatment and biodegradation. Appl Microbiol Biotechnol 51:883–890CrossRefPubMedGoogle Scholar
  26. Saifuddin N, Fazlili SA (2009) Effect of microwave and ultrasonic pretreatments on biogas production from anaerobic digestion of palm oil mill effluent. American Journal of Engineering and Applied Sciences 2:139–146CrossRefGoogle Scholar
  27. Salsabil MR, Prorot A, Casellas M, Dagot C (2009) Pretreatment effects of activated sludge: effect of sonication on aerobic and anaerobic digestibility. Chem Eng J 148:327–335CrossRefGoogle Scholar
  28. Salsabil MR, Laurent J, Casellas M, Dagot C (2010) Techno-economic evaluation of thermal treatment, ozonation and sonication for the reduction of wastewater biomass volume before aerobic or anaerobic digestion. J Hazard Mater 174:323–333CrossRefPubMedGoogle Scholar
  29. Sato S, Shibata C, Yazu M (1996) Nonthermal killing effect of microwave irradiation. Biotechnol Tech 10(3):145–150CrossRefGoogle Scholar
  30. Shamis Y, Taube A, Mitik-Dineva N, Croft R, Crawford RJ, Ivanova EP (2011) Specific electromagnetic effects of microwave radiation on Escherichia coli. Applied Environmental Microbiology 77(9):3017–3022PubMedCentralCrossRefPubMedGoogle Scholar
  31. Spencer RC, Hafiz S, Cook C (1985) Effect of microwave energy on the metabolism of Enterobacteriaceae. J Med Microbiol 19:269–272CrossRefPubMedGoogle Scholar
  32. Takatani S, Takayama S, Yamauchi T (1981) A study of anaerobic digestion for sewage sludge. Mitsubishi Juko Giho (Japan) 18:1–7Google Scholar
  33. Tiehm A, Nickel K, Neis U (1997) The use of ultrasound to accelerate the anaerobic digestion of sewage sludge. Water Science Technology 36(11):121–128CrossRefGoogle Scholar
  34. Tiehm A, Nickel K, Zellhorn M, Neis U (2001) Ultrasonic waste activated sludge disintegration for improving anaerobic stabilization. Water Res 35(8):2003–2009CrossRefPubMedGoogle Scholar
  35. Wahidunnabi AK, Eskicioglu C (2014) High pressure homogenization and two-phased anaerobic digestion for enhanced biogas conversion from municipal waste sludge. Water Res 66:430–446CrossRefPubMedGoogle Scholar
  36. Woo IS, Rhee IK, Park HD (2000) Differential damage in bacterial cells by microwave radiation on the basis of cell wall structure. Appl Environ Microbiol 66(5):2243–2247PubMedCentralCrossRefPubMedGoogle Scholar
  37. Yeneneh AM, Chong S, Sen TK, Ang HM, Kayaalp A (2013) Effect of ultrasonic, microwave and combined microwave-ultrasonic pretreatment of municipal sludge on anaerobic digester performance. Water Air and Soil Pollution 224(5):1–9CrossRefGoogle Scholar
  38. Yu Q, Lei HY, Li Z, Li HL, Chen K, Zhang XH, Liang RL (2010) Physical and chemical properties of waste-activated sludge after microwave treatment. Water Res 44:2841–2849CrossRefPubMedGoogle Scholar
  39. Zhang P, Zhang G, Wang W (2007) Ultrasonic treatment of biological sludge: floc disintegration, cell lysis and inactivation. Bioresour Technol 98(1):207–210CrossRefPubMedGoogle Scholar
  40. Zou J, Salminen WF, Roberts SM, Voellmy R (1998) Correlation between glutathione oxidation and trimerization of heat shock factor 1, an early step in stress induction of the Hsp response. Cell Stress and Chaperones 3(2):130–141PubMedCentralCrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Monica Angela Cella
    • 1
  • Deniz Akgul
    • 1
    • 2
  • Cigdem Eskicioglu
    • 1
    Email author
  1. 1.UBC Bioreactor Technology Group, School of EngineeringUniversity of British ColumbiaKelownaCanada
  2. 2.Department of Environmental EngineeringMarmara UniversityIstanbulTurkey

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