Abstract
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.
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References
Anzai Y, Kim H, Park J, Wakabayashi H, Oyaizu H (2000) Phylogenetic affiliation of the pseudomonads based on 16S rRNA sequence. Int J Syst Evol Microbiol 50:1563–1589
Bushnell H, Haas H (1941) The utilization of certain hydrocarbons by microörganisms. J Bacteriol 41:653–673
Curiale M (2000) MPN calculator, v. VB3. http://www.i2workout.com/mcuriale/mpn/index.html. Accessed 22 July 2010
Gibson DT, Hensley M, Yoshioka H, Mabry TJ (1970) Formation of (+)-cis-2, 3-dihydroxy-1-methylcyclohexa-4, 6-cyclohexadiene from toluene by Pseudomonas putida. Biochemistry 9:1626–1630. doi:10.1021/bi00809a023
Guicharnaud R, Arnalds O, Paton GI (2010) Short term changes of microbial processes in Icelandic soils to increasing temperatures. Biogeosciences 7:671–682. doi:10.5194/bg-7-671-2010
Haines JR, Wrenn BA, Holder EL, Strohmeier KL, Herrington RT, Venosa AD (1996) Measurement of hydrocarbon-degrading microbial population by a 96-well plate most-probable-number procedure. J Indust Microbiol 16:36–41
Hanzel J, Thullner M, Harms H, Wick LY (2011) Microbial growth with vapor-phase substrate. Environ Poll 159:858–864. doi:10.1016/j.envpol.2010.12.032
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
ISO 16703 (2004) Soil quality—Determination of content of hydrocarbon in the range C10 to C40 by gas chromatography. International Organization for Standardization, Geneva
Jeon CO, Park W, Padmanabhan P, DeRito C, Snape JR, Madsen EL (2003) Discovery of a bacterium, with distinctive dioxygenase, that is responsible for in situ biodegradation in contaminated sediment. PNAS 100:13591–13596. doi:10.1073/pnas.1735529100
Johnsen AR (2010) Introduction to microplate MPN-enumeration of hydrocarbon degraders. In: Timmis KN (ed) Handbook of hydrocarbon and lipid microbiology. Springer, Berlin, pp 4160–4172
Johnsen AR, Henriksen S (2009) Microplate MPN-enumeration of monocyclic- and dicyclic-aromatic hydrocarbon degraders via substrate phase-partitioning. Biodegradation 20:581–589. doi:10.1007/s10532-008-9236-9
Johnsen AR, Karlson U (2005) PAH degradation capacity of soil microbial communities—does it depend on PAH exposure? Microb Ecol 50:488–495. doi:10.1007/s00248-005-0022-5
Johnsen AR, Bendixen K, Karlson U (2002) Detection of microbial growth on polycyclic aromatic hydrocarbons in microtiter plates by using the respiration indicator WST-1. Appl Environ Microbiol 68:2683–2689. doi:10.1128/AEM.68.6.2683-2689.2002
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 103–109
Kirk JL, Klironomos JN, Lee H, Trevors JT (2005) The effects of perennial ryegrass and alfalfa on microbial abundance and diversity in petroleum contaminated soil. Environ Poll 133:455–465. doi:10.1016/j.envpol.2004.06.002
Kiyohara H, Nagao K (1978) The catabolism of phenanthrene and naphthalene by bacteria. J Gen Microbiol 105:69–75
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 1670
Mayer P, Fernqvist MM, Christensen PS, Karlson U, Trapp S (2007) Enhanced diffusion of polycyclic aromatic hydrocarbons in artificial and natural aqueous solutions. Environ Sci Technol 41:6148–6155. doi:10.1021/es070495t
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
Monod J (1949) The growth of bacterial cultures. Annual Rev Microbiol 3:371–394. doi:10.1146/annurev.mi.03.100149.002103
Pandey J, Chauhan A, Jain RK (2009) Integrative approaches for assessing the ecological sustainability of in situ bioremediation. FEMS Microbiol Rev 33:324–375. doi:10.1111/j.1574-6976.2008.00133.x
Piskonen R, Nyyssönen M, Rajamäki T, Itävaara M (2005) Monitoring of accelerated naphthalene-biodegradation in a bioaugmented soil slurry. Biodegradation 16:127–134. doi:10.1007/s10532-004-4893-9
Powell SM, Ferguson SH, Bowman JP, Snape I (2006) Using real-time PCR to assess changes in the hydrocarbon-degrading microbial community in Antarctic soil during bioremediation. Microb Ecol 52:523–532. doi:10.1007/s00248-006-9131-z
Pruesse E, Quast C, Knittel K, Fuchs BM, Ludwig W, Peplies J, Glöckner FO (2007) SILVA: a comprehensive online resource for quality checked and aligned ribosomal RNA sequence data compatible with ARB. Nucl Acids Res 35:7188–7196. doi:10.1093/nar/gkm864
Schloss PD (2009) A high-throughput DNA sequence aligner for microbial ecology studies. PLoS ONE 4:e8230. doi:10.1371/journal.pone.0008230
Schwarz FP, Wasik SP (1976) Fluorescence measurements of benzene, naphthalene, anthracene, pyrene, fluoranthene, and benzo[e]pyrene in water. Anal Chem 48:524–528
Shuttleworth KL, Cerniglia CE (1996) Bacterial degradation of low concentrations of phenanthrene and inhibition by naphthalene. Microb Ecol 31:305–317. doi:10.1007/BF00171574
Smith CJ, Osborn AM (2009) Advantages and limitations of quantitative PCR (Q-PCR)-based approaches in microbial ecology. FEMS Microbiol Ecol 67:6–20. doi:10.1111/j.1574-6941.2008.00629.x
Stieber M, Haeseler F, Werner P, Frimmel FH (1994) A rapid screening method for micro-organisms degrading polycyclic aromatic hydrocarbons in microplates. Appl Microbiol Biotechnol 40:753–755. doi:10.1007/BF00173340
Suzuki M, Rappe MS, Giovannoni SJ (1998) Kinetic bias in estimates of coastal picoplankton community structure obtained by measurements of small-subunit rRNA gene PCR amplicon heterogeneity. Appl Environ Microbiol 64:4522–4529
Tiirola MA, Suvilampi JE, Kulomaa MS, Rintala JA (2003) Microbial diversity in a thermophilic aerobic biofilm process: analysis by length heterogeneity PCR (LH-PCR). Water Res 37:2259–2268. doi:10.1016/S0043-1354(02)00631-0
Vance ED, Brooks PC, Jenkinson DS (1987) An extraction method for measuring soil microbial biomass C. Soil Biol Biochem 19:703–707. doi:10.1016/0038-0717(87)90052-6
Wallenius K, Rita H, Simpanen S, Mikkonen A, Niemi RM (2010) Sample storage for soil enzyme activity and bacterial community profiles. J Microbiol Methods 81:48–55. doi:10.1016/j.mimet.2010.01.021
Wrenn BA, Venosa AD (1996) Selective enumeration of aromatic and aliphatic hyrdocarbon degrading bacteria by a most-probable-number procedure. Can J Microbiol 42:252–258
Yagi JM, Madsen EL (2009) Diversity, abundance, and consistency of microbial oxygenase expression and biodegradation in a shallow contaminated aquifer. Appl Environ Microbiol 75:6478–6487. doi:10.1128/AEM.01091-09
Yu C, Chu K (2005) A quantitative assay for linking microbial community function and structure of a naphthalene-degrading microbial consortium. Environ Sci Technol 39:9611–9619. doi:10.1021/es051024e
Acknowledgments
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.
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10532_2011_9485_MOESM1_ESM.xls
Online Resource 1. Naphthalene concentration in uninoculated buffer as a function of distance from the spot source of naphthalene crystals (10 days exposure; 6 replicates). The measurement was performed by UV spectroscopy (280 nm) using a standard curve prepared from serial dilutions of saturated aqueous naphthalene solution. (XLS 26 kb)
10532_2011_9485_MOESM2_ESM.tif
Online Resource 2. Representative examples of the substrate plates (A: silicone oil method + INT, B: vapour method) and the negative control plates (C: silicone oil method + INT, D: vapour method) at the time when the results were scored. (TIFF 6025 kb)
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Wallenius, K., Lappi, K., Mikkonen, A. et al. Simplified MPN method for enumeration of soil naphthalene degraders using gaseous substrate. Biodegradation 23, 47–55 (2012). https://doi.org/10.1007/s10532-011-9485-x
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DOI: https://doi.org/10.1007/s10532-011-9485-x