Microorganisms are responsible for driving the biogeochemical cycling of elements on Earth. Despite their importance and vast diversity, the taxonomic identity of the microorganisms involved in any specific process has usually been confined to that small fraction of the microbiota that has been isolated and cultivated. The recent coupling of molecular biological methods with stable-isotope abundance in biomarkers has provided a cultivation-independent means of linking the identity of bacteria with their function in the environment. Here we show that 13C-DNA, produced during the growth of metabolically distinct microbial groups on a 13C-enriched carbon source, can be resolved from 12C-DNA by density-gradient centrifugation. DNA isolated from the target group of microorganisms can be characterized taxonomically and functionally by gene probing and sequence analysis. Application of this technique to investigate methanol-utilizing microorganisms in soil demonstrated the involvement of members of two phylogenetically distinct groups of eubacteria; the alpha-proteobacterial and Acidobacterium lineages. Stable-isotope probing thus offers a powerful new technique for identifying microorganisms that are actively involved in specific metabolic processes under conditions which approach those occurring in situ.
Well-drained non-agricultural soils mediate the oxidation of methane directly from the atmosphere, contributing 5 to 10% towards the global methane sink. Studies of methane oxidation kinetics in soil infer the activity of two methanotrophic populations: one that is only active at high methane concentrations (low affinity) and another that tolerates atmospheric levels of methane (high affinity). The activity of the latter has not been demonstrated by cultured laboratory strains of methanotrophs, leaving the microbiology of methane oxidation at atmospheric concentrations unclear. Here we describe a new pulse-chase experiment using long-term enrichment with 12CH4 followed by short-term exposure to 13CH4 to isotopically label methanotrophs in a soil from a temperate forest. Analysis of labelled phospholipid fatty acids (PLFAs) provided unambiguous evidence of methane assimilation at true atmospheric concentrations (1.8-3.6 p.p.m.v.). High proportions of 13C-labelled C18 fatty acids and the co-occurrence of a labelled, branched C17 fatty acid indicated that a new methanotroph, similar at the PLFA level to known type II methanotrophs, was the predominant soil micro-organism responsible for atmospheric methane oxidation.
Heightened concerns regarding the protection of terrestrial ecosystems at a national level has increased the need to develop a suite of indicators capable of assessing the quality, integrity and fertility of soils. Of the assays currently available, tests that measure aspects of soil function and associated parameters are among the most promising, since these integrate effects on soil quality at the highest level of organisation. In this study we describe results of the deployment of an indicator of soil functional integrity (the bait lamina test) that is designed to measure the feeding activity of soil invertebrates. Bait lamina was used at six grassland sites located along a transect from a smelter at Avonmouth (South-West England) used in the EU funded BIOPRINT II project. Results indicated highest bait removal (feeding) at sites furthest from the factory, intermediate feeding activity at intervening sites and extremely low activities at the two sites closest to the smelter. The strong decline in activity for the group of sites closest to the smelter corresponded with increasing metal concentrations suggesting a clear impact of metals on detritivorous invertebrate feeding. Comparisons of the results of the bait lamina study to previous invertebrate survey work suggested that the differences in observed bait removal can be attributed to direct effects of metals on the abundance and biodiversity of key decomposer groups such as earthworms, isopods, molluscs, myriapods, springtails and mites.
A stable mixed bacterial culture which degrades the herbicide linuron was isolated from soil by enrichment with linuron in a liquid mineral medium. Radio-respirometry studies showed that the culture mineralised linuron completely. No intermediate degradation products were detected in the medium. The culture was able to utilise linuron as a source of both nitrogen and carbon and was also able to degrade the related herbicides monolinuron and chlorbromuron and the possible intermediate degradation products of linuron: 3,4-dichlorophenyl-1methylurea, 3,4-dichlorophenylurea and 3,4-dichloroaniline. The culture was unable to degrade the 1,l-dimethyl substituted ureas monuron, diuron or metoxuron. The culture contained Gram-negative aerobic rods, and Grampositive aerobic non-spore-forming rods and cocco-bacilli. Of 124 ,isolates from the mixed culture, none degraded linuron in pure culture, indicating that a consortium of organisms is involved. Further investigation suggested that Pseudomonas spp. were important components of the population responsible for degradation.
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