Respiration of 13 C-Labeled Substrates Added to Soil in the Field and Subsequent 16S rRNA Gene Analysis of 13 C-Labeled Soil DNA

ABSTRACT Our goal was to develop a field soil biodegradation assay using 13 C-labeled compounds and identify the active microorganisms by analyzing 16S rRNA genes in soil-derived 13 C-labeled DNA. Our biodegradation approach sought to minimize microbiological artifacts caused by physical and/or nutritional disturbance of soil associated with sampling and laboratory incubation. The new field-based assay involved the release of 13 C-labeled compounds (glucose, phenol, caffeine, and naphthalene) to soil plots, installation of open-bottom glass chambers that covered the soil, and analysis of samples of headspace gases for 13 CO 2 respiration by gas chromatography/mass spectrometry (GC/MS). We verified that the GC/MS procedure was capable of assessing respiration of the four substrates added (50 ppm) to 5 g of soil in sealed laboratory incubations. Next, we determined background levels of 13 CO 2 emitted from naturally occurring soil organic matter to chambers inserted into our field soil test plots. We found that the conservative tracer, SF 6 , that was injected into the headspace rapidly diffused out of the soil chamber and thus would be of little value for computing the efficiency of retaining respired 13 CO 2 . Field respiration assays using all four compounds were completed. Background respiration from soil organic matter interfered with the documentation of in situ respiration of the slowly metabolized (caffeine) and sparingly soluble (naphthalene) compounds. Nonetheless, transient peaks of 13 CO 2 released in excess of background were found in glucose- and phenol-treated soil within 8 h. Cesium-chloride separation of 13 C-labeled soil DNA was followed by PCR amplification and sequencing of 16S rRNA genes from microbial populations involved with 13 C-substrate metabolism. A total of 29 full sequences revealed that active populations included relatives of Arthrobacter , Pseudomonas , Acinetobacter , Massilia , Flavobacterium , and Pedobacter spp. for glucose; Pseudomonas , Pantoea , Acinetobacter , Enterobacter , Stenotrophomonas , and Alcaligenes spp. for phenol; Pseudomonas , Acinetobacter , and Variovorax spp. for naphthalene; and Acinetobacter , Enterobacter , Stenotrophomonas , and Pantoea spp. for caffeine.