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Applied and Environmental Microbiology, June 2006, p. 3901-3907, Vol. 72, No. 6
0099-2240/06/$08.00+0     doi:10.1128/AEM.02779-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Estimating High-Affinity Methanotrophic Bacterial Biomass, Growth, and Turnover in Soil by Phospholipid Fatty Acid 13C Labeling

P. J. Maxfield,1 E. R. C. Hornibrook,2 and R. P. Evershed1*

Organic Geochemistry Unit, Bristol Biogeochemistry Research Centre, School of Chemistry, University of Bristol, Cantock's Close, Bristol BS8 1TS, United Kingdom,1 Department of Earth Sciences, Bristol Biogeochemistry Research Centre, University of Bristol, Wills Memorial Building, Queen's Road, Bristol BS8 1RJ, United Kingdom2

Received 24 November 2005/ Accepted 20 March 2006

A time series phospholipid fatty acid (PLFA) 13C-labeling study was undertaken to determine methanotrophic taxon, calculate methanotrophic biomass, and assess carbon recycling in an upland brown earth soil from Bronydd Mawr (Wales, United Kingdom). Laboratory incubations of soils were performed at ambient CH4 concentrations using synthetic air containing 2 parts per million of volume of 13CH4. Flowthrough chambers maintained a stable CH4 concentration throughout the 11-week incubation. Soils were analyzed at weekly intervals by gas chromatography (GC), GC-mass spectrometry, and GC-combustion-isotope ratio mass spectrometry to identify and quantify individual PLFAs and trace the incorporation of 13C label into the microbial biomass. Incorporation of the 13C label was seen throughout the experiment, with the rate of incorporation decreasing after 9 weeks. The {delta}13C values of individual PLFAs showed that 13C label was incorporated into different components to various extents and at various rates, reflecting the diversity of PLFA sources. Quantitative assessments of 13C-labeled PLFAs showed that the methanotrophic population was of constant structure throughout the experiment. The dominant 13C-labeled PLFA was 18:1{omega}7c, with 16:1{omega}5 present at lower abundance, suggesting the presence of novel type II methanotrophs. The biomass of methane-oxidizing bacteria at optimum labeling was estimated to be about 7.2 x 106 cells g–1 of soil (dry weight). While recycling of 13C label from the methanotrophic biomass must occur, it is a slower process than initial 13CH4 incorporation, with only about 5 to 10% of 13C-labeled PLFAs reflecting this process. Thus, 13C-labeled PLFA distributions determined at any time point during 13CH4 incubation can be used for chemotaxonomic assessments, although extended incubations are required to achieve optimum 13C labeling for methanotrophic biomass determinations.

* Corresponding author. Mailing address: Bristol Biogeochemistry Research Centre, School of Chemistry, University of Bristol, Cantock's Close, Bristol BS8 1TS, United Kingdom. Phone: 44 117 928 7671. Fax: 44 117 929 3746. E-mail: r.p.evershed{at}bris.ac.uk.

Applied and Environmental Microbiology, June 2006, p. 3901-3907, Vol. 72, No. 6
0099-2240/06/$08.00+0     doi:10.1128/AEM.02779-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • McDonald, I. R., Bodrossy, L., Chen, Y., Murrell, J. C. (2008). Molecular Ecology Techniques for the Study of Aerobic Methanotrophs. Appl. Environ. Microbiol. 74: 1305-1315 [Full Text]  


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