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Method for measuring substrate preferences by individual members of microbial consortia proposed for bioaugmentation

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Abstract

In this study we used the assimilation of isotope labeled CO2 to measure the substrate preferences by two different bioaugmentation mixtures proposed for bioremediation of diesel oil contamination. All active microorganisms assimilate CO2 in various carboxylation processes involved in growth. The CO2 assimilation by the two mixtures was measured upon addition of glucose, diesel oil or specific compounds present in diesel oil (naphthalene, toluene, hexadecane, and octane). It was shown that within short term incubations with diesel oil (<5 h), one bioaugmentation mixture was superior to the other regarding the assimilation of CO2. This observation was confirmed in a labor-intensive long term microcosm study (60 days). The applied method open various possibilities for fast pre-testing of substrate-preferences by microbial-bioaugmentation mixtures without microcosm experiments, onsite tests, and complicated chemical analysis. This study also demonstrates the possibility to obtain further information on the substrate preferences at a single cell level of phylogenetically defined microbial subgroups in bioaugmentation mixtures, based on combined analyses of microautoradiography and fluorescence in situ hybridization.

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Abbreviations

AI:

CO2 assimilation Index

BS and JR:

Microbial-bioaugmentation mixtures tested in this study (see material and methods)

FISH:

Fluorescence in situ hybridization

HetCO2-MAR:

Microautoradiography of heterotrophic bacteria on the basis of assimilation of 14CO2

MAR:

Microautoradiography

References

  • Alexander M (1995) How toxic are toxic chemicals in soil? Environ Sci Technol 29:2713–2717

    Article  CAS  Google Scholar 

  • Amann R, Binder BJ, Olson RJ, Chisholm SW, Devereux R, Stahl DA (1990) Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analysing mixed microbial populations. Appl Environ Microbiol 56:1919–1925

    PubMed  CAS  Google Scholar 

  • Amann R, Snaidr J, Wagner M, Ludwig W, Schleifer KH (1996) In situ visualization of high genetic diversity in a natural microbial community. J Bacteriol 178:3496–3500

    PubMed  CAS  Google Scholar 

  • Barker HA (1941) The chemistry and metabolism of bacteria. Annu Rev Biochem 10:553–580

    Article  CAS  Google Scholar 

  • Bouchez T, Patureau D, Dabert P, Juretschko S, Dore J, Delgenes P, Moletta R, Wagner M (2000) Ecological study of a bioaugmentation failure. Environ Microbiol 2:179–190

    Article  PubMed  CAS  Google Scholar 

  • Burggraf S, Mayer T, Amann R, Schadhauser S, Woese CR, Stetter KO (1994) Identifying members of the domain Archaea with rRNA-targeted oligonucleotide probes. Appl Environ Microbiol 60:3112–3119

    PubMed  CAS  Google Scholar 

  • Daims H, Bruhl A, Amann R, Schleifer KH, Wagner M (1999) The domain-specific probe EUB338 is insufficient for the detection of all Bacteria: Development and evaluation of a more comprehensive probe set. Syst Appl Microbiol 22:434–444

    PubMed  CAS  Google Scholar 

  • Daims H, Nielsen JL, Nielsen PH, Schleifer KH, Wagner M (2001) In situ characterization of Nitrospira-like nitrite oxidizing bacteria active in wastewater treatment plants. Appl Environ Microbiol 67:5273–5284

    Article  PubMed  CAS  Google Scholar 

  • Dedysh SN, Liesack W, Khmelenina VN, Suzina NE, Trotsenko YA, Semrau JD, Bares AM, Panikov NS, Tiedje JM (2000) Methylocella palustris gen. nov., sp nov., a new methane-oxidizing acidophilic bacterium from peat bags, representing a novel subtype of serine-pathway methanotrophs. Int J Syst Evol Microbiol 50:955–969

    PubMed  CAS  Google Scholar 

  • Dijkhuizen L, Harder W (1985) Microbial metabolism of carbon dioxide. In: Dalton H (Eds), Comprehensive biotechnology: the principles of biotechnology, vol 1. Pergamon Press Ltd., Oxford, pp 409–423

    Google Scholar 

  • Donaldson JM, Henderson GS (1989) A dilute medium to determine population size of ammonium oxidizers in forest soils. Soil Sci Soc Am J 53:1608–1611

    Article  CAS  Google Scholar 

  • Doronina NV, Trotsenko YA (1984) Levels of carbondioxide assimilation in bacteria with different pathways of C1 metabolism. Mikrobiologiya 53:885–889

    CAS  Google Scholar 

  • El Fantroussi S, Agathos SN (2005) Is bioaugmentation a feasible strategy for pollutant removal and site remediation? Curr Opin Microbiol 8:268–275

    Article  PubMed  CAS  Google Scholar 

  • Gentry TJ, Rensing C, Pepper IL (2004) New approaches for bioaugmentation as a remediation technology. Crit Rev Environ Sci Technol 34:447–494

    Article  CAS  Google Scholar 

  • Gray ND, Howarth R, Pickup RW, Jones JG, Head IM (1999) Substrate uptake by uncultured bacteria from the genus Achromatium determined by microautoradiography. Appl Environ Microbiol 65:5100–5106

    PubMed  CAS  Google Scholar 

  • Grossmann S (1994) Bacterial activity in sea-ice and open water of the weddell sea, Antarctica- a microautoradiographic study. Microb Ecol 28:1–18

    Article  CAS  Google Scholar 

  • Hesselsoe M, Brandt KK, Sørensen J (2001) Quantification of ammonia-oxidizing bacteria in soil using microcolony technique combined with fluorescent in situ hybridization (FISH). FEMS Microbiol Ecol 38:87–95

    CAS  Google Scholar 

  • Hesselsoe M, Nielsen JL, Roslev P, Nielsen PH (2005) Isotope labeling and microautoradiography of active heterotrophic bacteria on the basis of assimilation of 14CO2. Appl Environ Microbiol 71:646–655

    Article  PubMed  CAS  Google Scholar 

  • Johnson BT, Romanenko VI (1984) Xenobiotic pertubation of microbial growth as measured by CO2 uptake in aquatic heterotrophic bacteria. J Great Lakes Res 10:245–250

    Article  CAS  Google Scholar 

  • Loy A, Horn M, Wagner M (2003) probeBase- an online resource for rRNA-targeted oligonucleotide probes. Nucleic Acids Res 31:514–516

    Article  PubMed  CAS  Google Scholar 

  • MacDonald RM, Spokes JR (1980) A selective diagnostic medium for ammonia-oxidizing bacteria. FEMS Microbiol Lett 8:143–145

    Article  CAS  Google Scholar 

  • Major DW, McMaster ML, Cox EE, Edwards EA, Dworatzek SM, Hendrickson ER, Starr MG, Payne JA, Buonamici LW (2002) Field demonstration of successful bioaugmentation to achieve dechlorination of tetrachloroethene to ethene. Environ Sci Technol 36:5106–5116

    Article  PubMed  CAS  Google Scholar 

  • Manz W, Amann R, Ludwig W, Wagner M, Schleifer KH (1992) Phylogenetic oligodeoxynucleotide probes for major subclasses of proteobacteria: problems and solutions. Syst Appl Microbiol 15:593–600

    Google Scholar 

  • Meier H, Amann R, Ludwig W, Schleifer KH (1999) Specific oligonucleotide probes for in situ detection of a major group of Gram-positive bacteria with low DNA G + C content. Syst Appl Microbiol 22:186–196

    PubMed  CAS  Google Scholar 

  • Meyer-Reil LA (1978) Autoradiography and epifluorescence microscopy combined for the determination of number and spectrum of actively metabolizing bacteria in natural waters. Appl Environ Microbiol 36:506–512

    PubMed  CAS  Google Scholar 

  • Miethling R, Karlson U (1996) Accelerated mineralization of pentachlorophenol in soil upon inoculation with Mycobacterium chlorophenolicum PCP1 and Sphingomonas chlorophenolica RA2. Appl Environ Microbiol 62:4361–4366

    PubMed  CAS  Google Scholar 

  • Mueller JG, Resnick SM, Shelton ME, Pritchard PH (1992) Effect of inoculation on the biodegradation of weathered Prudhoe Bay crude oil. J Ind Microbiol 10:95–102

    Article  Google Scholar 

  • Neef A (1997) Anwendung der in situ Einzelzell-Identifizierung von Bakterien zur Populationsanalyse in komplexen mikrobiellen Biozönosen. Ph.D. thesis from the Technical University of Munich, Germany

  • Neef A, Amann R, Schlesner H, Schleifer KH (1998) Monitoring a widespread bacterial group: in situ detection of planctomycetes with 16S rRNA-targeted probes. Microbiology 144:3257–3266

    PubMed  CAS  Google Scholar 

  • Nielsen JL, Christensen D, Kloppenborg M, Nielsen PH (2003) Quantification of cell-specific substrate uptake by probe- defined bacteria under in situ conditions by microautoradiography and fluorescence in situ hybridization. Environ Microbiol 5:202–211

    Article  PubMed  CAS  Google Scholar 

  • Nielsen JL, Nielsen PH (2002) Enumeration of acetate-consuming bacteria by microautoradiography under oxygen and nitrate respiring conditions in activated sludge. Water Res 36:421–428

    Article  PubMed  CAS  Google Scholar 

  • Nielsen JL, Nielsen PH (2005) Advances in microscopy: microautoradiography of single cells. Method Enzymol 397:237–256

    Article  CAS  Google Scholar 

  • Paerl HW, Steppe TF, Reid RP (2001) Bacterially mediated precipitation in marine stromatolites. Environ Microbiol 3:123–130

    Article  PubMed  CAS  Google Scholar 

  • Roller C, Wagner M, Amann R, Ludwig W, Schleifer KH (1994) In situ probing of Gram-positive bacteria with high DNA G + C content using 23S rRNA- targeted oligonucleotides. Microbiology 140:2849–2858

    PubMed  CAS  Google Scholar 

  • Romanenko VI (1961) Use of the autoradiographic method for a quantitative assay of methane-oxidizing bacteria. Mikrobiologiya 30:292–293

    Google Scholar 

  • Romanenko VI (1964) Heterotrophic assimilation of CO2 by bacterial flora of water. Mikrobiologiya 33:610–614

    Google Scholar 

  • Roslev P, Larsen MB, Jørgensen D, Hesselsoe M (2004) Use of heterotrophic CO2 as a measure og metabolic activity in planctonic and sessile cells. J Microbiol Methods

  • Roslev P, Madsen PL, Thyme JB, Henriksen K (1998) Degradation of phthalate and di-(-2-ethylhexyl)phtalate by indigenous and inoculated microorganisms in sludge-amended soil. Appl Environ Microbiol 64:4711–4719

    PubMed  CAS  Google Scholar 

  • Rousseaux S, Hartmann A, Lagacherie B, Piutti S, Andreux F, Soulas G (2002) Inoculation of an atrazine-degrading strain, Chelatobacter heintzii Cit1, in four different soils: effects of different inoculum densities. Chemosphere 51:569–576

    Article  CAS  Google Scholar 

  • Salanitro JP, Johnson PC, Spinnler GE, Maner PM, Wisniewski HL, Bruce C (2000) Field-scale demonstration of enhanced MTBE bioremediation through aquifer bioaugmentation and oxygenation. Environ Sci Technol 34:4152–4162

    Article  CAS  Google Scholar 

  • Saralov AI, Krylova IN, Kuznetsov SI (1984) A modification of Sorokin´s method for proportional determination of bacterial chemosynthesis and organotrophic CO2 assimilation in lakes. Mikrobiologiya 53:989–996

    CAS  Google Scholar 

  • Sengeløv G, Sørensen S, Frette L, Kroer N (2000) A new method for determining the metabolic activity of specific bacterial populations in soil using tritiated leucine and immunomagnetic separation. Biol Fertil Soils 32:202–208

    Article  Google Scholar 

  • Smith AE, Hristova K, Wood I, Mackay D, Lory E, Lorenzana D, Scow KM (2005) Comparison of biostimulation versus bioaugmentation with bacterial strain PM1 for treatment of groundwater contaminated with methyl tertiary butyl ether (MTBE). Environ Health Perspect 113:1–9

    Google Scholar 

  • Stahl DA, Amann R (1991) Development and application of nucleic acid probes. In: Stackebradt E, Goodfellow M (eds), Nucleic acid techniques in bacterial systematics. John Wiley & Sons, Chichester, England, pp 205–248

    Google Scholar 

  • Tabor PS, Neihof RA (1984) Direct determination of activities for microorganisms of chesapeake bay populations. Appl Environ Microbiol 48:1012–1019

    PubMed  CAS  Google Scholar 

  • van Veen JA, van Overbeek LS, van Elsas JD (1997) Fate and activity of microorganisms introduced into soil. Microbiol Mol Biol Rev 61:121–135

    PubMed  Google Scholar 

  • Werkman CH, Wood HG (1942) On the metabolism of bacteria. Bot Rev VIII:1–68

    Article  Google Scholar 

  • Yang YR, Zarda A, Zeyer J (2003) Combination of microautoradiography and fluorescence in situ hybridization for identification of microorganisms degrading xenobiotic contaminants. Environ Toxicol Chem 22:2840–2844

    Article  PubMed  CAS  Google Scholar 

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Acknowledgements

The principal funding for this study was provided by Danish Technical Research Council (grants # 26-03-0036 to MH and #26-03-02250 to JLN). Additional generous private support to MH from the R98-foundation is highly appreciated. Maria Grøn and Pia Jensen are acknowledged for their contribution to the microcosm work (M.Sc. thesis at the Department of biotechnology, chemistry and environmental engineering, Aalborg University).

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  1. Martin Hesselsoe

    Present address: Amphi Consult, Aalborg, Denmark

Authors and Affiliations

  1. Department of biotechnology, chemistry and environmental engineering, Aalborg University Denmark, Aalborg, Denmark

    Martin Hesselsoe, Marianne L. Bjerring, Kaj Henriksen & Jeppe L. Nielsen

  2. DMR A/S, Jerslev, Denmark

    Per Loll

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  1. Martin Hesselsoe
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  2. Marianne L. Bjerring
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Correspondence to Martin Hesselsoe.

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Hesselsoe, M., Bjerring, M.L., Henriksen, K. et al. Method for measuring substrate preferences by individual members of microbial consortia proposed for bioaugmentation. Biodegradation 19, 621–633 (2008). https://doi.org/10.1007/s10532-007-9167-x

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  • DOI: https://doi.org/10.1007/s10532-007-9167-x

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