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

Abstract

A time series phospholipid fatty acid (PLFA) ¹³ C-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 CH ₄ concentrations using synthetic air containing 2 parts per million of volume of ¹³ CH ₄ . Flowthrough chambers maintained a stable CH ₄ 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 ¹³ C label into the microbial biomass. Incorporation of the ¹³ C label was seen throughout the experiment, with the rate of incorporation decreasing after 9 weeks. The δ ¹³ C values of individual PLFAs showed that ¹³ C label was incorporated into different components to various extents and at various rates, reflecting the diversity of PLFA sources. Quantitative assessments of ¹³ C-labeled PLFAs showed that the methanotrophic population was of constant structure throughout the experiment. The dominant ¹³ C-labeled PLFA was 18:1ω7c, with 16:1ω5 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 × 10 ⁶ cells g ⁻¹ of soil (dry weight). While recycling of ¹³ C label from the methanotrophic biomass must occur, it is a slower process than initial ¹³ CH ₄ incorporation, with only about 5 to 10% of ¹³ C-labeled PLFAs reflecting this process. Thus, ¹³ C-labeled PLFA distributions determined at any time point during ¹³ CH ₄ incubation can be used for chemotaxonomic assessments, although extended incubations are required to achieve optimum ¹³ C labeling for methanotrophic biomass determinations.