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Applied and Environmental Microbiology, February 2007, p. 798-807, Vol. 73, No. 3
0099-2240/07/$08.00+0     doi:10.1128/AEM.01491-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Nutrient Amendments in Soil DNA Stable Isotope Probing Experiments Reduce the Observed Methanotroph Diversity{triangledown} ,{dagger}

Aurélie Cébron,1,{ddagger} Levente Bodrossy,2 Nancy Stralis-Pavese,2 Andrew C. Singer,3 Ian P. Thompson,3 James I. Prosser,4 and J. Colin Murrell1*

Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, United Kingdom,1 Department of Bioresources, Division of Life and Environmental Sciences, ARC Seibersdorf Research GmbH, A-2444 Seibersdorf, Austria,2 Environmental Biotechnology Section, NERC Centre for Ecology and Hydrology—Oxford, Mansfield Road, Oxford OX1 3SR, United Kingdom,3 School of Biological Sciences, Cruickshank Building, St. Machar Drive, Aberdeen AB24 3UU, Scotland, United Kingdom4

Received 28 June 2006/ Accepted 14 November 2006

Stable isotope probing (SIP) can be used to analyze the active bacterial populations involved in a process by incorporating 13C-labeled substrate into cellular components such as DNA. Relatively long incubation times are often used with laboratory microcosms in order to incorporate sufficient 13C into the DNA of the target organisms. Addition of nutrients can be used to accelerate the processes. However, unnatural concentrations of nutrients may artificially change bacterial diversity and activity. In this study, methanotroph activity and diversity in soil was examined during the consumption of 13CH4 with three DNA-SIP experiments, using microcosms with natural field soil water conditions, the addition of water, and the addition of mineral salts solution. Methanotroph population diversity was studied by targeting 16S rRNA and pmoA genes. Clone library analyses, denaturing gradient gel electrophoresis fingerprinting, and pmoA microarray hybridization analyses were carried out. Most methanotroph diversity (type I and type II methanotrophs) was observed in nonamended SIP microcosms. Although this treatment probably best reflected the in situ environmental conditions, one major disadvantage of this incubation was that the incorporation of 13CH4 was slow and some cross-feeding of 13C occurred, thereby leading to labeling of nonmethanotroph microorganisms. Conversely, microcosms supplemented with mineral salts medium exhibited rapid consumption of 13CH4, resulting in the labeling of a less diverse population of only type I methanotrophs. DNA-SIP incubations using water-amended microcosms yielded faster incorporation of 13C into active methanotrophs while avoiding the cross-feeding of 13C.

* Corresponding author. Mailing address: Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, United Kingdom. Phone: 44-24-76523553. Fax: 44-24-76523568. E-mail: j.c.murrell{at}warwick.ac.uk.

{triangledown} Published ahead of print on 12 November 2006.

{dagger} Supplemental material for this article may be found at http://aem.asm.org/.

{ddagger} Present address: LIMOS, UHP Nancy 1, Faculté des Sciences, B.P. 239, F 54506, Vandoeuvre les Nancy, France.

Applied and Environmental Microbiology, February 2007, p. 798-807, Vol. 73, No. 3
0099-2240/07/$08.00+0     doi:10.1128/AEM.01491-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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