Simplified MPN method for enumeration of soil naphthalene degraders using gaseous substrate
We describe a simplified microplate most-probable-number (MPN) procedure to quantify the bacterial naphthalene degrader population in soil samples. In this method, the sole substrate naphthalene is dosed passively via gaseous phase to liquid medium and the detection of growth is based on the automated measurement of turbidity using an absorbance reader. The performance of the new method was evaluated by comparison with a recently introduced method in which the substrate is dissolved in inert silicone oil and added individually to each well, and the results are scored visually using a respiration indicator dye. Oil-contaminated industrial soil showed slightly but significantly higher MPN estimate with our method than with the reference method. This suggests that gaseous naphthalene was dissolved in an adequate concentration to support the growth of naphthalene degraders without being too toxic. The dosing of substrate via gaseous phase notably reduced the work load and risk of contamination. The result scoring by absorbance measurement was objective and more reliable than measurement with indicator dye, and it also enabled further analysis of cultures. Several bacterial genera were identified by cloning and sequencing of 16S rRNA genes from the MPN wells incubated in the presence of gaseous naphthalene. In addition, the applicability of the simplified MPN method was demonstrated by a significant positive correlation between the level of oil contamination and the number of naphthalene degraders detected in soil.
KeywordsHydrocarbon degraders MPN Naphthalene Optical density Phase partitioning Soil
This work was funded by the Academy of Finland, University of Helsinki and Ekokem Oy. We thank Elina Kondo, University of Helsinki, for supervision in the oil analytics and Rannveig Guicharnaud, Agricultural University of Iceland, for supervision in the microbial biomass measurements.
- Curiale M (2000) MPN calculator, v. VB3. http://www.i2workout.com/mcuriale/mpn/index.html. Accessed 22 July 2010
- Huang WE, Ferguson A, Singer AC, Lawson K, Thompson IP, Kalin RM, Larkin MJ, Bailey MJ, Whiteley AS (2009) Resolving genetic functions within microbial populations: in situ analyses using rRNA and mRNA stable isotope probing coupled with single-cell raman-fluorescence in situ hybridization. Appl Environ Microbiol 75:234–241. doi: 10.1128/AEM.01861-08 PubMedCrossRefGoogle Scholar
- ISO 16703 (2004) Soil quality—Determination of content of hydrocarbon in the range C10 to C40 by gas chromatography. International Organization for Standardization, GenevaGoogle Scholar
- Johnsen AR (2010) Introduction to microplate MPN-enumeration of hydrocarbon degraders. In: Timmis KN (ed) Handbook of hydrocarbon and lipid microbiology. Springer, Berlin, pp 4160–4172Google Scholar
- Jørgensen K, Järvinen O, Sainio P, Salminen J, Suortti A (2005) Quantification of soil contamination, hydrocarbons in the range C10 to C40. In: Margesin R, Schinner F (eds) Manual of soil analysis: monitoring and assessing soil bioremediation. Springer, Berlin, pp 103–109Google Scholar
- Kiyohara H, Nagao K (1978) The catabolism of phenanthrene and naphthalene by bacteria. J Gen Microbiol 105:69–75Google Scholar
- Lauderdale TL, Chapin KC, Murray PR (1999) Reagents, stains and media. In: Murray PR, Baron EJ, Pfaller MA, Tenover FC, Yolken RH (eds) Manual of clinical microbiology, 7th edn. American Society for Microbiology, Washington, p 1670Google Scholar
- Mikkonen A, Kondo E, Lappi K, Wallenius K, Lindström K, Hartikainen H, Suominen L (2010) Contaminant and plant derived changes in soil chemical and microbiological indicators during fuel oil rhizoremediation with Galega orientalis. Geoderma 160:336–346. doi: 10.1016/j.geoderma.2010.10.001 CrossRefGoogle Scholar
- Schwarz FP, Wasik SP (1976) Fluorescence measurements of benzene, naphthalene, anthracene, pyrene, fluoranthene, and benzo[e]pyrene in water. Anal Chem 48:524–528Google Scholar