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 Clabeled 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.Achieving a mechanistic understanding of microorganisms where they dwell, in terrestrial and aquatic field habitats, is one of the major goals of microbial ecology; such understanding is facilitated by an ability to directly measure microbial metabolic processes and to identify microorganisms responsible for particular field biogeochemical reactions (4,24,35,41,48). But a variety of methodological obstacles have traditionally prevented investigators from simultaneously documenting identity and activity in real-world habitats such as soil. The most notable obstacles are the size discrepancy between humans and microorganisms, incomplete understanding of microhabitat physicochemical characteristics, a large reservoir of inactive, but potentially responsive cells in environmental samples, and the related propensity for microbial comm...

Importance of soil organic matter for the diversity of microorganisms involved in the degradation of organic pollutants

“…Equally, Arthrobacter and Rhodococcus isolates can degrade phenol Karigar et al, 2006;Martinkova et al, 2009), and members of both genera were detected by DNA-SIP as phenanthrene degraders in soil (Cebron et al, 2011). Another DNA-SIP study with phenol retrieved mainly Gammaproteobacteria from a silt loam soil (Padmanabhan et al, 2003). The diverging results suggest that soil and environmental properties have a strong effect on the diversity of bacteria involved in phenol degradation.…”

Bacterial genome replication at subzero temperatures in permafrost

“…These 12 C and 13 C-DNA bands were collected by pipette, rather than fractionating the entire gradient. This approach has been shown to reduce contamination and ensure clean 12 C-control amplifications (Padmanabhan et al, 2003;Gallagher et al, 2005;Kerkhof et al, 2011). This does not imply that 12 C DNA contamination is not present throughout the gradient.…”

Halobacteriovorax, an underestimated predator on bacteria: potential impact relative to viruses on bacterial mortality

“…To monitor predation by Halobacteriovorax by molecular methods, DNA was extracted from 100-μl aliquots of water from the experimental flask (see Supplementary Information). Then, 13 C DNA was separated from unlabeled DNA by density gradient centrifugation (Padmanabhan et al, 2003) and was amplified using both a universal primer GM5F (5´-CCTACGGGAGGCAGCAG-3´) with a GC clamp attached to its 5′ end and 907R (5′-CCGT CAATTCCTTTGAGTTT-3′) (Muyzer et al, 1995) and BALO-specific primers, Bac-676F (5′-ATTT CGCATGTAGGGGTA-3′) and Bac-1442R (5′-GCCA CGGCTTCAGGTAAG-3′). Amplicons were run on a denaturing gradient gel electrophoresis (DGGE) gel gradient as described in Supplementary Information to characterize bacterial communities in the experimental flasks.…”