Enumeration of methanotrophic bacteria in the cover soil of an aged municipal landfill.

The enumeration of methanotrophic bacteria in the cover soil of an aged municipal landfill was carried out using (1) fluorescent in situ hybridization (FISH) with horseradish peroxidase-labeled oligonucleotide probes and tyramide signal amplification, also known as catalyzed reporter deposition-FISH (CARD-FISH), and (2) most probable number (MPN) method. The number of methanotrophs was determined in cover soil samples collected during April-November 2003 from a point with low CH(4) emission. The number of types I and II methanotrophs obtained by CARD-FISH varied from 15 +/- 2 to 56 +/- 7 x 10(8) cells g(-1) absolute dry mass (adm) of soil and methanotrophs of type I dominated over type II. The average number of methanotrophs throughout the cover soil profile was highest during May-September when the cover soil temperature was above 13 degrees C. Methanotrophs accounted for about 50% of the total bacterial population in the deepest cover soil layer owing to higher availability of substrate (CH(4)). A lower number of methanotrophs (7 x 10(2) to 17 x 10(5) cells g(-1) adm of soil) was determined by the MPN method compared to the CARD-FISH counts, thus confirming previous results that the MPN method is limited to the estimation of the culturable species that can be grown under the incubation conditions used. The number of culturable methanotrophs correlated with the methane-oxidizing activity measured in laboratory assays. In comparison to the incubation-based measurements, the number of methanotrophs determined by CARD-FISH better reflected the actual characteristics of the environment, such as release and uptake of CH(4), temperature, and moisture, and availability of substrates.