Estimating high-affinity methanotrophic bacterial biomass, growth, and turnover in soil by phospholipid fatty acid 13C labeling

A time series phospholipid fatty acid (PLFA) C-13-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 (CH4)-C-13. 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 C-13 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 C-13 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 C-13-labeled PLFAs showed that the methanotrophic population was of constant structure throughout the experiment. The dominant C-13-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 10(6) 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 (CH4)-C-13 incorporation, with only about 5 to 10% of C-13-labeled PLFAs reflecting this process. Thus, C-13-labeled PLFA distributions determined at any time point during 13 CH, incubation can be used for chemotaxonomic assessments, although extended incubations are required to achieve optimum 13C labeling for methanotrophic biomass determinations.