Applied Microbiology and Biotechnology

, Volume 81, Issue 5, pp 977–985

Impact of cell density on microbially induced stable isotope fractionation

  • Makeba Kampara
  • Martin Thullner
  • Hauke Harms
  • Lukas Y. Wick
Environmental Biotechnology


Quantification of microbial contaminant biodegradation based on stable isotope fractionation analysis (SIFA) relies on known, invariable isotope fractionation factors. The microbially induced isotope fractionation is caused by the preferential cleavage of bonds containing light rather than heavy isotopes. However, a number of non-isotopically sensitive steps preceding the isotopically sensitive bond cleavage may affect the reaction kinetics of a degradation process and reduce the observed (i.e., the macroscopically detectable) isotope fractionation. This introduces uncertainty to the use of isotope fractionation for the quantification of microbial degradation processes. Here, we report on the influence of bacterial cell density on observed stable isotope fractionation. Batch biodegradation experiments were performed under non-growth conditions to quantify the toluene hydrogen isotope fractionation by exposing Pseudomonas putida mt-2(pWWO) at varying cell densities to different concentrations of toluene. Observed isotope fractionation depended significantly on the cell density. When the cell density rose from 5 × 105 to 5 × 108cells/mL, the observed isotope fractionation declined by 70% and went along with a 55% decrease of the degradation rates of individual cells. Theoretical estimates showed that uptake-driven diffusion to individual cells depended on cell density via the overlap of the cells’ diffusion-controlled boundary layers. Our data suggest that biomass effects on SIFA have to be considered even in well-mixed systems such as the cell suspensions used in this study.


Bioavailability Cell density Biodegradation Fractionation factor Stable isotope fractionation analysis Toluene 


  1. Boone DR, Johnson RL, Liu Y (1989) Diffusion of the interspecies electron carriers H2 and formate in methanogenic ecosystems and its implications in the measurement of Km for H2 or formate uptake. Appl Environ Microbiol 55:1735–1741Google Scholar
  2. Born M, Dorr H, Levin I, Munnich KO (1988) Methane concentration in aerated soils in West-Germany. Chem Geol 70:101–101CrossRefGoogle Scholar
  3. Bosma TNP, Middeldorp PJM, Schraa G, Zehnder AJB (1997) Mass transfer limitation of biotransformation: quantifying bioavailability. Environ Sci Technol 31:248–252CrossRefGoogle Scholar
  4. Braunlich M, Aballanin O, Marik T, Jockel P, Brenninkmeijer CAM, Chappellaz J, Barnola JM, Mulvaney R, Sturges WT (2001) Changes in the global atmospheric methane budget over the last decades inferred from 13C and D isotopic analysis of Antarctic firn air. J Geophys Res Atmos 106:20465–20481CrossRefGoogle Scholar
  5. Calder JA, Parker PL (1973) Geochemical implications of induced changes in C-13 Fractionation by blue-green algae. Geochim Cosmochim Acta 37:133–140CrossRefGoogle Scholar
  6. Chartrand MMG, Waller A, Mattes TE, Elsner M, Lacrampe-Couloume G, Gossett JM, Edwards EA, Sherwood Lollar B (2005) Carbon isotopic fractionation during aerobic vinyl chloride degradation. Environ Sci Technol 39:1064–1070CrossRefGoogle Scholar
  7. Cichocka D, Siegert M, Imfeld G, Andert J, Beck K, Diekert G, Richnow HH, Nijenhuis I (2007) Factors controlling the carbon isotope fractionation of tetra- and trichloroethene during reductiove dechlorination by Sulfurospirillum ssp. and Desulfitobacterium sp. strain PCE-S. FEMS Microbiol Ecol 62:98–107CrossRefGoogle Scholar
  8. Duetz WA, Wind B, van Andel JG, Barnes MR, Williams PA, Rutgers M (1998) Biodegradation kinetics of toluene, m-xylene, p-xylene and their intermediates through the upper TOL pathway in Pseudomonas putida (pWW0). Microbiol SGM 144:1669–1675CrossRefGoogle Scholar
  9. Elsner M, Zwank L, Hunkeler D, Schwarzenbach RP (2005) A new concept linking observable stable isotope fractionation to transformation pathways of organic pollutants. Environ Sci Technol 39:6896–6916CrossRefGoogle Scholar
  10. Griebler C, Safinowski M, Vieth A, Richnow HH, Meckenstock RU (2004) Combined application of stable carbon isotope analysis and specific metabolites determination for assessing in situ degradation of aromatic hydrocarbons in a tar oil-contaminated aquifer. Environ Sci Technol 38:617–631CrossRefGoogle Scholar
  11. Gupta M, Tyler S, Cicerone R (1996) Modeling atmospheric delta (CH4)-13C and the causes of recent changes in atmospheric CH4 amounts. J Geophys Res-Atmos 101:22923–22932CrossRefGoogle Scholar
  12. Harms H, Zehnder AJB (1994) Influence of substrate diffusion on degradation of dibenzofuran and 3-chlorodibenzofuran by attached and suspended bacteria. Appl Environ Microbiol 60:2736–2745Google Scholar
  13. Hunkeler D, Anderson N, Aravena R, Bernasconi SM, Butler BJ (2001) Hydrogen and carbon isotope fractionation during aerobic biodegradation of benzene. Environ Sci Technol 35:3462–3467CrossRefGoogle Scholar
  14. Kampara M, Thullner M, Harms H, Wick LY (2008) Impact of bioavailability restrictions on microbially induced stable isotope fractionation: 2. Experimental evidence. Environ Sci Technol 42:6552–6558Google Scholar
  15. Mancini SA, Hirschorn SK, Elsner M, Lacrampe-Couloume G, Sleep BE, Edwards EA, Sherwood Lollar B (2006) Effects of trace element concentration on enzyme controlled stable isotope fractionation during aerobic biodegradation of toluene. Environ Sci Technol 40:7675–7681CrossRefGoogle Scholar
  16. March JG, Pringle CM (2003) Food web structure and basal resource utilization along a tropical island stream continuum, Puerto Rico. Biotropica 35:84–93Google Scholar
  17. Mariotti A, Germon JC, Hubert P, Kaiser P, Letolle R, Tardieux A, Tardieux P (1981) Experimental determination of nitrogen kinetic isotope fractionation—some principles—illustration for the denitrification and nitrification processes. Plant Soil 62:413–430CrossRefGoogle Scholar
  18. Meckenstock RU, Morasch B, Griebler C, Richnow HH (2004) Stable isotope fractionation analysis as a tool to monitor biodegradation in contaminated acquifers. J Contam Hydrol 75:215–255CrossRefGoogle Scholar
  19. Morasch B, Richnow HH, Schink B, Meckenstock RU (2001) Stable hydrogen and carbon isotope fractionation during microbial toluene degradation: mechanistic and environmental aspects. Appl Environ Microbiol 67:4842–4849CrossRefGoogle Scholar
  20. Nijenhuis I, Andert J, Beck K, Kastner M, Diekert G, Richnow HH (2005) Stable isotope fractionation of tetrachloroethene during reductive dechlorination by Sulfurospirillum multivorans and Desulfitobacterium sp strain PCE-S and abiotic reactions with cyanocobalamin. Appl Environ Microbiol 71:3413–3419CrossRefGoogle Scholar
  21. Northrop DB (1981) The Expression of isotope effects on enzyme-catalyzed reactions. Annu Rev Biochem 50:103–131CrossRefGoogle Scholar
  22. O'Leary MH (1980) Carbon isotope fractionation in plants. Phytochem 20:553–567CrossRefGoogle Scholar
  23. Pardue JW, Scalan RS, Vanbaalen C, Parker PL (1976) Maximum carbon isotope fractionation in photosynthesis by blue-green algae and a green algae. Geochim Cosmochim Acta 40:309–312CrossRefGoogle Scholar
  24. Park R, Epstein S (1960) Carbon isotope fractionation during photosynthesis. Geochim Cosmochim Ac 21:110–126CrossRefGoogle Scholar
  25. Rayleigh JSW (1896) Theoretical considerations respecting the separation of gases by diffusion and similar processes. Philos Mag 42:493–498Google Scholar
  26. Schmidt TC, Zwank L, Elsner M, Berg M, Meckenstock RU, Haderlein SB (2004) Compound-specific stable isotope analysis of organic contaminants in natural environments: a critical review of the state of the art, prospects, and future challenges. Anal Bioanal Chem 378:283–300CrossRefGoogle Scholar
  27. Shaw JP, Harayama S (1992) Purification and characterization of the NADH-acceptor reductase component of xylene monooxygenase encoded by the TOL plasmid pWW0 of Pseudomonas putida mt2. Eur J Biochem 209:51–61CrossRefGoogle Scholar
  28. Staal M, Thar R, Kuhl M, van Loosdrecht MCM, Wolf G, de Brouwer JFC, Rijstenbil JW (2007) Different carbon isotope fractionation patterns during the development of phototrophic freshwater and marine biofilms. Biogeosci 4:613–626CrossRefGoogle Scholar
  29. Templeton AS, Chu KH, Alvarez-Cohen L, Conrad ME (2006) Variable carbon isotope fractionation expressed by aerobic CH4-oxidizing bacteria. Geochim Cosmochim Acta 70:1739–1752CrossRefGoogle Scholar
  30. Thullner M, Kampara M, Harms H, Wick LY (2008) Impact of bioavailability restrictions on microbially induced stable isotope fractionation: 1. Theoretical calculations. Environ Sci Technol 42:6544–6551Google Scholar
  31. Tyler SC, Crill PM, Brailsford GW (1994) 13C/12C fractionation of methane during oxidation in a temperate forested soil. Geochim Cosmochim Acta 58:1625–1633CrossRefGoogle Scholar
  32. van Leeuwen HP, Köster W (2004) Physicochemical kinetics and transport at biointerfaces: setting the stage. In: van Leeuwen HP, Koester W(eds) Physicochemical kinetics and transport at chemical-biological interphases. IUPAC series in analytical and physical chemistry of environmental systems. Wiley, Chichester, pp 401–444Google Scholar
  33. Whiticar MJ (1990) A geochemical perspective of natural gas and atmospheric methane. Org Geochem 16:531–547CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2008

Authors and Affiliations

  • Makeba Kampara
    • 1
  • Martin Thullner
    • 1
  • Hauke Harms
    • 1
  • Lukas Y. Wick
    • 1
  1. 1.Department of Environmental MicrobiologyUFZ—Helmholtz Centre for Environmental ResearchLeipzigGermany

Personalised recommendations