Methods of assessing microbial activity and inhibition under anaerobic conditions: a literature review


This work reviews the existing methodologies for assessing microbial activity and inhibition under anaerobic conditions. The anaerobic digestion process consists of several metabolic steps–the Anaerobic Digestion Model No. 1 (ADM1) has attempted to describe these steps in the form of a mathematical model with the intention of providing a reference base for all further efforts in the modelling of anaerobic processes. The existence of a reference point for modelling has highlighted the fact that there is a lack of coherence between the many different methodologies for experimentally assessing anaerobic activity and inhibition.

A working group of the International Water Association was recently founded to harmonise the existing methodologies with the ultimate intention of developing a unified reference procedure– a primary objective of the group will be the establishment of a standard terminology in the field of anaerobic digestion, activity and inhibition assessment. Secondly, it will compare the existing methodologies and develop standard protocols for assessing the kinetic parameters (e.g. maximum uptake rate, half-saturation constant) of anaerobic processes that may be entered directly into ADM1 and its successors.

This paper revises and enlarges a contribution presented by the authors at the workshop “Harmonisation of anaerobic biodegradation, activity and inhibition assays” (Ligthart & Nieman 2002, Proc. workshop held in Orta (Italy) June 7–8, 2002) and aims to promote a clear understanding of the currently established methodology.

Numerous methods have been developed over the past 30 years, since Van den Berg et al. (1974, Biotechnol Bioeng 16(11)– 1459–1469) measured methanogenic activity, by using a manometric device equipped with a photoelectric sensor to quantify the gas production. Methanogenesis is often the rate limiting step of the entire process and since the quantification of gas flowrate is relatively easy to perform, most of the methods reported in literature monitor the production of biogas. These methods can be termed volumetric or manometric methods, as the volume of biogas produced or the pressure increase due to gas production inside a close vessel are assessed, respectively. However, this same concept can be employed to assess activity or inhibition of individual metabolic steps preceding the methanogenic one, providing that they are rate limiting for the whole process. The reliability of activity assessment through gas measurement has been proven to be strongly dependent on the equilibrium between liquid and gas phase in a closed vessel. This can be influenced by many factors, e.g. the amount and characteristics of the test substrate; the concentration of the biomass; the gas-to-liquid ratio– all these aspects will need to be addressed in the standard procedure. Other direct or indirect methods, targeting physico-chemical or microbiological parameter exist and have been investigated by many authors. Besides the interest for research purposes, the definition of reference methods to assess activity and inhibition can be of great interest for engineers, both phy. Specific reference procedures might be needed for particular applications, e.g. the (kinetic) study of rate limiting microbial steps and might require ad-hoc methodologies to be devised. A microbiological technique such as FISH, coupled with microsensors have been reported to have a great potential in the near future.


activity anaerobic inhibition methods 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Amann, R, Kühl, M 1998In situ methods for assessment of microorganisms and their activitiesCurr. Opin. Microbiol.1352358Google Scholar
  2. Amann, R, Fuchs, BM, Behrens, S 2001The identification of microorganisms by fluorescence in situ hybridisationCurr. Opinion Biotechnol.12231236Google Scholar
  3. Anderson, GK, Yang, G 1992Determination of bicarbonate and total volatile acid concentration in anaerobic digesters using a simple titrationWat. Environ. Res.64(1)5359Google Scholar
  4. Angelidaki, I, Schmidt, JE, Ellegaard, L, Ahring, BK 1998An automated system for simultaneous monitoring of gas evolution in multiple closed vesselsJ. Microbiol. Meth.3393100Google Scholar
  5. Angenent, LT, Sung, S, Raskin, L 2002Methanogenic population dynamics during startup of a full-scale anaerobic sequencing batch reactor treating swine wasteWat. Res.3646484654Google Scholar
  6. Annweiler, E, Materna, A, Safinowski, M, Kappler, A, Richnow, HH, Michaelis, W, Meckenstock, RU 2000Anaerobic degradation of 2-methylnaphthalene by a sulfate-reducing enrichment cultureAppl. Environ. Microbiol.6653295333Google Scholar
  7. ASTM d 5210 (1992) Determining the anaerobic biodegradation of plastic materials in the presence of municipal sewage sludgeGoogle Scholar
  8. Batstone, DJ, Keller, J, Angelidaki, RI, Kalyuzhnyi, SV, Pavlostathis, SG, Rozzi, A, Sanders, WTM, Siegrist, H, Vavilin, VA 2002Anaerobic Digestion Model No 1 (ADM1) Scientific and Technical Report No. 13 IWA Task Group for Mathematical Modelling of Anaerobic WastewaterIWA PublishingLondon88Google Scholar
  9. Battersby, NS 2000The biodegradability and microbial toxicity testing of lubricants–some recommendationsChemosphere4110111027Google Scholar
  10. Beaubien, A, Jolicoeur, C, Alary, JF 1989Automated high sensitivity gas metering system for biological processesJ. Environ. Eng.118551567Google Scholar
  11. Bhupathiraju, VK, Hernandez, MT, Landfear, D, Alvarez-Cohen, L 1999Application of a tetrazolium dye as an indicator of viability in anaerobic bacteriaJ. Microbiol. Methods37231243Google Scholar
  12. Burland, SM, Edwards, EA 1999Anaerobic benzene biodegradation linked to nitrate reductionAppl. Environ. Microbiol.65529533Google Scholar
  13. Campos, CMM, Chernicaro, CAL 1991The use of the SMA-test for measuring toxicity in anaerobic sludgesWat. Sci. Technol.24103111Google Scholar
  14. Chen, CC, Lin, CY 2003Using sucrose as a substrate in an anaerobic hydrogen-producing reactorAdv. Environ. Res.7695699Google Scholar
  15. Chung, Y-C, Neethling, JB 1988ATP as a measure of anaerobic sludge digester activityJ. Wat. Pollut. Contr. Fed.60108112Google Scholar
  16. Chung, Y-C, Neethling, JB 1989Microbial activity measurements for anaerobic sludge digestionJ. Wat. Pollut. Contr. Fed.61343349Google Scholar
  17. Coates, JD, Coughlan, MF, Colleran, E 1996Simple method for the measurement of the hydrogenotrophic methanogenic activity of anaerobic sludgesJ. Microbiol. Meth.2623746Google Scholar
  18. Cohen, A 1992Effects of some industrial chemicals on anaerobic activity measured by sequential automated methanometryWat. Sci. Tech.251120Google Scholar
  19. Concannon, F, Reynolds, PJ, Hennigan, A, Colleran, E 1988Automated measurement of the specific methanogenic activity of anaerobic digestion biomassBiochem. Soc. Trans.17425Google Scholar
  20. Long, EF, Wickham, GS, Pace, NR 1989Phylogenetic stain: ribosomal RNA-based probes for the identification of single microbial cellsScience24313601363Google Scholar
  21. de Zeeuw W & Lettinga G (1980) Acclimation of digested sewage sludge during start-up of upflow anaerobic sludge blanket (UASB) reactor. In: Proc. 35th Ind. Wastewater Conf., Purdue University, LafayetteGoogle Scholar
  22. Delbès, C, Moletta, R, Godon, J-J 2001Bacterial and archaeal 16S rDNA and 16S rRNA dynamics during an acetate crisis in an anaerobic digestor ecosystemFEMS Microbiol. Ecol.351926Google Scholar
  23. Dolfing, J, Mulder, J-W 1985Comparison of methane production rate and coenzyme F420 content in anaerobic granular sludgeAppl. Environ. Microbiol.4911421145Google Scholar
  24. ECETOC (1988) Guideline for screening of chemical for anaerobic biodegradability. Technical Report 28, Brussels.Google Scholar
  25. Fang, HHP, Jia, X-S 1999Formation of interim by-products in methanogenic degradation of butyrateWat. Res.3317911798Google Scholar
  26. Fdz.Polanco, F, Fdz.Polanco, M, Fernandez, N, Urueña, MA, Garcia, PA, Villaverde, S 2001New process for simultaneous removal of nitrogen and sulphate under anaerobic conditionsWat. Res.3511111114Google Scholar
  27. Feitkenhauer, H, von Sachs, J, Meyer, U 2002On-line titration of volatile fatty acids for the process control of anaerobic digestion plantsWat. Res.36212218Google Scholar
  28. García, MT, Campos, E, Sánchez-Leal, J, Ribosa, I 2000Anaerobic degradation and toxicity of commercial cationic surfactants in anaerobic screening testsChemosphere41705710Google Scholar
  29. Glauser, M, Jenny, B, Aragno, M 1984An inexpensive, automatic gas meter for laboratory-scale methane digesters and other gas-evolving systemsJ. Microbiol. Meth.2159164Google Scholar
  30. Gonzalez-Gil, G, Kleerebezem, R, Lettinga, G 2002Assessment of metabolic properties and kinetic parameters of methanogenic sludge by on-line methane production rate measurementsAppl. Microbiol. Biotechnol.58248254Google Scholar
  31. Gorris, LG, Kok, TM, Kroon, BM, Drift, C, Vogels, GD 1988Relationship between methanogenic cofactor content and maximum specific methanogenic activity of anaerobic granular sludgesAppl. Environ. Microbiol.5411261130Google Scholar
  32. Gruden, CL, Fevig, S, Abu-Dalo, M, Hernandez, M 20035-Cyano-2,3-ditolyl tetrazolium chloride (CTC) reduction in a mesophilic anaerobic digester: measuring redox behavior, differentiating abiotic reduction, and comparing FISH response as an activity indicatorJ. Microbiol. Meth.525968Google Scholar
  33. Guwy, AJ, Hawkes, DL, Hawkes, FR, Rozzi, A 1994Characterisation of a prototype industrial on-line analyser for bicarbonate/carbonate monitoringBiotechnol. Bioeng.4413251330Google Scholar
  34. Häggblom, MM, Knight, VK, Kerkhof, LJ 2000Anaerobic decomposition of halogenated aromatic compoundsEnviron. Pollut.107199207Google Scholar
  35. Hills, DJ, Nakano, K 1984Effect of particle size on anaerobic digestion of tomato solid wasteAgric. Wastes10285295Google Scholar
  36. Hongwei, Y, Zhanpeng, J, Shaoqi, S, Tang, WZ 2002INT-dehydrogenase activity test for assessing anaerobic biodegradability of organic compoundsEcotoxicol. Environ. Saf.53416421Google Scholar
  37. ISO 11734 (1995) Water quality–Evaluation of the ultimate anaerobic biodegradability of organic compounds in digested sludge–method by measurement of the biogas production. International Standard (First Edition 15-12-1995)Google Scholar
  38. ISO/DIS 14853 (1997) Evaluation of ultimate anaerobic biodegradation of plastic materials in an aqueous system–method by analysis of carbon conversion to carbon dioxide and methaneGoogle Scholar
  39. James, A, Chernicaro, CAL, Campos, CMM 1990The development of a new methodology for the assessment of specific methanogenic activityWat. Res.24813825Google Scholar
  40. Jolicoeur, C, To, T, Beaubien, A 1998Flow microcalorimetry in monitoring biological activity of aerobic and anaerobic wastewater treatment processesAnal. Chim. Acta213165176Google Scholar
  41. Jörg, R 1999Anaerobe, biologische Abbaubarkeit in aquatischen Screening-Tests unter Berücksichtigung von Untersuchung mit dem neuen Meßgerät MethanomatPh.D. Thesis. ISWA Universität StuttgartGermanyGoogle Scholar
  42. Kida, K, Shigematsu, T, Kijima, J, Numaguchi, M, Mochinaga, Y, Abe, N Morimura S 2001Influence of Ni2+ and Co2+ on methanogenic activity and the amounts of coenzymes involved in methanogenesisJ. Biosci. Bioeng.91590595Google Scholar
  43. Kuroda, K, Silveira, RG, Nishio, N, Sunahara, H, Nagai, S 1991Measurement of dissolved hydrogen in an anaerobic digestion process by a membrane-covered electrodeJ. Ferment. Technol.71418423Google Scholar
  44. Kuss ML & Young JC (1992) Apparatus for measuring gas flow using bubble volume. U.S. Patent No. 5,092,181 (March 3, 1992)Google Scholar
  45. Lee, Y, Takashima, M, Speece, RE 1987Microcomputer pH control of multiple bioreactorsJ. Biotechnol. Bioeng.30329330Google Scholar
  46. Ligthart J & Nieman H (2002) Harmonisation of anaerobic biodegradation, activity and inhibition assays. Proc. Workshop held in Orta (Italy) June 7–8, 2002Google Scholar
  47. Logan, BE, Oh, S-E, Kim, IS, Ginkel, S 2002Biological hydrogen production measured in batch anaerobic respirometersEnviron. Sci. Technol.3625302535Google Scholar
  48. Lovley, DR, Woodward, JC, Chapelle, FH 1996Rapid anaerobic benzene oxidation with a variety of chelated Fe(III) formsAppl. Environ. Microbiol.62288291Google Scholar
  49. Madsen, T, Rasmussen, HB 1996A method for screening the potential toxicity of organic chemicals to methanogenic gas productionWat. Sci. Technol.33213220Google Scholar
  50. Marison, IW, Liu, JS, Ampuero, S, Von Stockar, U, Schenker, B 1998Biological reaction calorimetry: Development of high sensitivity bio-calorimetersThermochim. Acta30157173Google Scholar
  51. Mata-Alvarez, J, Martinez-Viturtia, A, Torres, R 1986A simple device to measure biogas production in laboratory scale digesterBiotechnol. Lett.8719720Google Scholar
  52. Meyer, B, Heinzle, E 1998Dynamic determination of anaerobic acetate kinetics using membrane mass spectrometryBiotechnol. Bioeng.57127135Google Scholar
  53. Moletta, R, Albagnac, G 1982A gas meter for low rates of flow: Application to the methane fermentationBiotechnol. Lett.4319322Google Scholar
  54. Mösche, M, Jördening, HJ 1999Comparison of different models of substrate and product inhibition in anaerobic digestionWat. Res.3325452554Google Scholar
  55. Müller W-R and Frommert I (2002) Existing standardised methods for anaerobic biodegradability testing. In: Ligthart and Nieman (2002)Google Scholar
  56. Owen, WF, Stuckey, DC, Healy, JB, Young, LY, Mc Carty, PL 1979Bioassay for monitoring biochemical methane potential and anaerobic toxicityWat. Res.13485492Google Scholar
  57. Painter, HA, Reynolds, P, Comber, S 2003Application of the headspace CO2 method (ISO 14593) to the assessment of the ultimate biodegradability of surfactants: Results of a calibration exerciseChemosphere502938Google Scholar
  58. Pavlostathis, SG, Giraldo-Gomez, E 1991Kinetics of anaerobic treatment: A review, CritRev. Environ. Control21411490Google Scholar
  59. Pelz, O, Chatzinotas, A, Zarda-Hess, A, Abraham, W-R, Zeyer, J 2001Tracing toluene-assimilating sulphate-reducing bacteria using 13C-incorporation in fatty acids and whole-cell hybridisationFEMS Microbiol. Ecol.38123131Google Scholar
  60. Pollice, A, Rozzi, A, Tomei, MC, Di Pinto, AC, Limoni, N 2000Monitoring the inhibitory effects of NaCl on anaerobic wastewater treatment processes by the Rantox biosensorEnviron. Technol.21535544Google Scholar
  61. Pollice, A, Rozzi, A, Tomei, MC, Di Pinto, AC, Laera, G 2001Inhibiting effects of chloroform on anaerobic microbial consortia as monitored by the Rantox biosensorWat. Res.3511791190Google Scholar
  62. Puhar, E, Einsele, A, Buehler, H, Ingold, W 1980Steam sterilisable pCO2 electrodeBiotechnol. Bioeng.2224112416Google Scholar
  63. Raskin, L, Poulsen, LK, Noguera, DR, Rittmann, BE, Stahl, DA 1994Quantification of methanogenic groups in anaerobic biological reactors by oligonucleotide probe hybridizationAppl. Environ. Microbiol.6012411248Google Scholar
  64. Raskin, L, Amann, RI, Poulsen, LK, Rittmann, BE, Stahl, DA 1995Use of ribosomal RNA-based molecular probes for characterisation of complex microbial communities in anaerobic biofilmsWat. Sci. Technol.31261272Google Scholar
  65. Remigi, E eds. 2001Biodegradability and activity assessment under anaerobic conditionsPh.D. Thesis. Politecnico di MilanoItalyGoogle Scholar
  66. Remigi E, Rozzi A & Fux C (2002) Assessment of microbial kinetics (aceticlastic methanogenesis and Anammox) by a titration bioassay. Proc. VII Taller y Seminario Latinoamericano sobre Digestion Anaerobia, Mérida (Mexico) 22–25 OctoberGoogle Scholar
  67. Rozzi A, Brunetti A, Palmisano V & Stella P (1983) Automatic data acquisition and processing system for acetoclastic methanogenic activity tests. Proc. Anaerobic wastewater treatment symposium (Noordwijkerhout, The Netherlands, November 1983)Google Scholar
  68. Rozzi, A, Tomei, MC, Di, Pinto AC, Limoni, N 1997Monitoring toxicity in anaerobic digesters by the Rantox biosensor: Theoretical backgroundBiotechnol. Bioeng.553340Google Scholar
  69. Rozzi, A, Tomei, MC, Di Pinto, AC, Limoni, N 1999Monitoring toxicity in anaerobic digesters by the Rantox biosensor: Calibration testsBiores. Technol.68155163Google Scholar
  70. Rozzi A & Ficara E (2001) Titration biosensors for risk assessment of contaminated sites and groundwater. Proc. 1st Workshop SENSPOL: Sensing Technologies for contaminated sites on groundwater. University of Alcalà (Spain), 9–11 MayGoogle Scholar
  71. Rozzi, A, Remigi, E, Buckley, C 2001Methanogenic activity measurements by the MAIA biosensor: Instruction guideWat. Sci. Technol.44287294Google Scholar
  72. Rozzi, A, Castellazzi, L, Speece, RE 2002Acetoclastic methanogenic activity measurement by a titration bioassayBiotech. Bioeng.772026Google Scholar
  73. Sanders, WTM 2001Anaerobic hydrolysis during digestion of complex substratesPh.D. Thesis. Wageningen UniversityThe NetherlandsGoogle Scholar
  74. Sanders, WTM, Zeeman, G, Lettinga, G 2002Hydrolysis kinetics of dissolved polymer substratesWat. Sci. Technol.4599104Google Scholar
  75. Schonberg, JC, Bhattacharya, SK, Madura, RL, Mason, SH, Conway, RA 1997Evaluation of anaerobic treatment of selected petrochemical wastesJ. Hazardous Mat.544763Google Scholar
  76. Shelton, EA, Tiedje, JM 1984General method for determining anaerobic biodegradation potentialAppl. Environ. Microbiol.47850857Google Scholar
  77. South, CR, Hogsett, DAL, Lynd, DLR 1995Modelling simultaneous saccharification and fermentation of lignocellulose to ethanol in batch and continuous reactorsEnzyme Microbiol. Technol.17797803Google Scholar
  78. Speece, RE 1996Anaerobic biotechnology for industrial wastewatersArchae PressNashville (TN, USA)Google Scholar
  79. Sponza, DT 2003Toxicity and treatability of carbontetrachloride and tetrachloroethylene in anaerobic batch culturesInt. Biodeterior. Biodegr.51119127Google Scholar
  80. Strous, M, Kuenen, JG, Jetten, MSM 1999Key physiology of anaerobic ammonium oxidationAppl. Environ. Microbiol.6532483250Google Scholar
  81. Stumm, W, Morgan, J 1996Aquatic Chemistry: Chemical Equilibria and Rates in Natural WatersWiley InterscienceNew York, USAGoogle Scholar
  82. Suflita, JM, Concannon, F 1995Screening test for assessing the anaerobic biodegradation of pollutant chemicals in subsurface environmentsJ. Microbiol. Meth.21267281Google Scholar
  83. Takashima, M, Speece, RE 1989Mineral nutrient requirements for high-rate methane fermentation of acetate at low SRTRes. J. Wat. Poll. Contr. Fed.6116451650Google Scholar
  84. Tipton FK (1992) Principles of enzyme assays and kinetic studies in enzyme assays. In: Eseinthal R & Ranson MJ (Ed.) A Practical Approach, Oxford University Press, OxfordGoogle Scholar
  85. Umbreit, WW, Burris, RH, Stayffer, JF 1964Manometric TechniquesBurgessBerks4th edn.Google Scholar
  86. Valcke, D, Verstraete, W 1983A practical method to estimate the acetoclastic methanogenic biomass in anaerobic sludgesJ. Wat. Poll. Contr. Fed.5511911195Google Scholar
  87. Van, den Berg L, Lentz, CP, Athey, RJ, Rooke, EA 1974Assessment of methanogenic activity in anaerobic digestion apparatus and methodBiotechnol. Bioeng.1614591469Google Scholar
  88. Veeken, A, Hamelers, B 1999Effect of temperature on the hydrolysis rate of selected biowaste componentsBiores. Technol.69249255Google Scholar
  89. Veiga, MC, Soto, M, Mendez, R, Lema, JM 1990A new device for measurement control of gas production by bench scale anaerobic digesterWat. Res.2415511554Google Scholar
  90. Vidal, G, Soto, M, Field, J, Mendéz-Pampín, R, Lema, JM 1997Anaerobic biodegradability and toxicity of wastewater from chlorine and total chlorine-free bleaching of eucalyptus kraft pulpsWat. Res.3124872494Google Scholar
  91. Voolapalli, RK, Stuckey, DC 2001Hydrogen production in anaerobic reactors during shock loads–influence of formate production and H2 kineticsWat. Res.3518311841Google Scholar
  92. Yang, J, Speece, RE 1986The effects of chloroform toxicity on methane fermentationWat. Res.2012731279Google Scholar
  93. Young, JC, Tabak, HH 1993Multilevel protocol for assessing the fate and effect of toxic organic chemicals in anaerobic treatment processesWat. Environ. Res.65(1)3435Google Scholar

Copyright information

© Kluwer Academic Publishers 2004

Authors and Affiliations

  1. 1.DIIAR, Politecnico di MilanoMilanoItaly
  2. 2.Pollution Research GroupUniversity of KwaZulu NatalDurbanSouth Africa

Personalised recommendations