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...

Thirty-nine xylanolytic bacteria tentatively identified as Clostridia were isolated from a selection of agricultural and forest soil samples, and water–sediment–fibre samples from acidic springs in the Yellowstone National Park. Screening for xylan hydrolysis was performed using an enriched agar medium at pH 5.5 with Remazol–xylan as the indicator, with or without an initial enrichment using xylan as the major carbon source. From 13 of the most highly xylanolytic strains, 9 were tentatively identified as Clostridium acetobutylicum, 2 as Clostridium butyricum, and 2 as Clostridium beijerinckii. The C. acetobutylicum isolate 3BYR utilized 80% of oat spelt xylan as a carbon source during growth. The bacterium exhibited very high extracellular xylanase and xylosidase activities and, as well, α-L-arabinofuranosidase and α-glucuronidase activities. Glucuronidase activity was documented by the release of 4-O-methyl-α-D-glucuronic acid from birchwood xylan. The results of this work indicate the ubiquity of xylanolytic Clostridia, and that the previously unreported activity, α-glucuronidase, has been demonstrated in C. acetobutylicum. Key words: α-glucuronidase, xylanase, Clostridium acetobutylicum, xylan.