Stable-isotope probing is a method used in microbial ecology that provides a means by which specific functional groups of organisms that incorporate particular substrates are identified without the prerequisite of cultivation. Stable-isotope-labeled carbon (13C) or nitrogen (15N) sources are assimilated into microbial biomass of environmental samples. Separation and molecular analysis of labeled nucleic acids (DNA or RNA) reveals phylogenetic and functional information about the microorganisms responsible for the metabolism of a particular substrate. Here, we highlight general guidelines for incubating environmental samples with labeled substrate and provide a detailed protocol for separating labeled DNA from unlabeled community DNA. The protocol includes a modification of existing published methods, which maximizes the recovery of labeled DNA from CsCl gradients. The separation of DNA and retrieval of unlabeled and labeled fractions can be performed in 4-5 days, with much of the time being committed to the ultracentrifugation step.

Temporal and spatial variation of soil bacterial communities was evaluated with both molecular and metabolic profiling techniques. Soil cores (20 cm deep) were taken from an upland grassland in the Scottish Borders (UK) over 3 days in July 1999, and on single days in October 1999, April 2000, and August 2000. Cores were separated into four 5-cm depths to examine vertical spatial distribution. The 0-5-, 5-10- and 10-15-cm samples represented organic horizons whilst the 15-20-cm depths were from a mineral horizon. The potential metabolic activities were analysed using BIOLOG-GN plates, whereas genotypic diversity was evaluated using molecular profiling of amplified 16S rRNA and 16S rDNA gene fragments (denaturing gradient gel electrophoresis (DGGE)). BIOLOG-GN analysis revealed decreased substrate utilisation in the lowest depths, which was coupled with changes in the DNA and RNA DGGE profiles. Seasonal variation was pronounced in the 5-10-cm and 10-15-cm organic horizons for the July samplings whilst the 15-20-cm depths appeared more stable. Potential factors influencing the observed changes in bacterial communities resulting from soil depth and sampling time are discussed.

1. Global interest in building healthy soils combined with new DNA sequencing technologies has led to the generation of a vast amount of soil microbial community (SMC) data. 2. SMC analysis is being adopted widely for monitoring ecological restoration trajectories. However, despite the large and growing quantity of soil microbial data, it remains unclear how these data inform and best guide restoration practice. 3. Here, we examine assumptions around SMC as a tool for guiding ecosystem restoration and evaluate the effectiveness of using species inventories of SMC as a benchmark for restoration success. 4. We investigate other approaches of assessing soil health, and conclude that we can significantly enhance the utility of species inventory data for ecological restoration by complementing it with the use of non-molecular approaches.

More than half of the fertilizer applied to farmers' field is lost, causing significant economic losses. To overcome this, a polymer-coated rock mineral fertilizer was investigated using wheat (Triticum aestivum L. cv. Wyalkatchem). In addition, a multispecies microbial inoculant was added to seeds as a biostimulant to enhance fertilizer use efficiency. Thus, this glasshouse experiment investigated the effect of polymer-coated rock mineral fertilizer with or without a multispecies microbial inoculant on wheat growth in a sandy soil. We hypothesized that the polymer-coated rock mineral fertilizer combined with a microbial inoculant would be more effective than non-coated fertilizer at increasing growth, nutrient uptake and yield of wheat in sandy soil. Both the polymer-coated and non-coated rock mineral fertilizer, either with or without microbial seed inoculation, increased shoot growth at tillering and maturity but root growth only increased at maturity. Grain yield did not differ between the fertilizer treatments except that they were lower for the non-coated rock mineral fertilizer when combined with microbial inoculation. In the absence of microbial inoculation, soil amended with polymercoated fertilizer had lower residual soil P and K. The hypothesis that microbial inoculation would improve the growth, nutrient uptake and yield of wheat was not supported in this 2 experiment. Sequencing of 16S rRNA identified Proteobacteria and Actinobacteria as the key phyla in rhizosphere soil. Fertilizer treatments altered alpha diversity (OTU richness, Inverse Simpson and Fisher indices) but had no effect on evenness. This polymer-coated rock mineral fertilizer has potential to substitute for or complement more soluble fertilizers, but there was no benefit of inclusion of the multispecies microbial inoculant on plant growth or yield.

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The restoration of vegetation post-mining is particularly challenging in extreme conditions such as Mediterranean systems where soil moisture is limiting, soil temperature fluctuates dramatically, and soil carbon is very low. In such systems, soil microbial communities may play an important role in attenuating extreme conditions. Thus, vegetation establishment on such sites may be curtailed by depauperate soil communities. Soil fungal communities, in particular, are essential for nutrient turn over but we know very little about how these communities respond to mining and post-mining restoration. Fungi may be significantly affected by restoration practices. For example, the inclusion of deeper soil profiles (i.e., "overburden") into restoration events is rare, but may expedite fungal community development. We studied a successional gradient of sand mine restoration in a former Banksia woodland in SW Australia to determine whether soil fungal communities recovered after 13 years. We also asked whether the inclusion of overburden into restoration sites improved soil fungal community development. Overall, fungal communities did not return to a pre-disturbance state by 13 years, nor did the inclusion of overburden affect their trajectory. Longer term studies are need to determine when, if ever, fungal communities are restored, and what effect this has nascent vegetation.

Environmental factors relating to soil pH are widely known to be important in structuring soil bacterial communities, yet the relationship between taxonomic community composition and functional diversity remains to be determined. Here, we analyze geographically distributed soils spanning a wide pH gradient and assess the functional gene capacity within those communities using whole genome metagenomics. Low pH soils consistently had fewer taxa (lower alpha and gamma diversity), but only marginal reductions in functional alpha diversity and equivalent functional gamma diversity. However, coherent changes in the relative abundances of annotated genes between pH classes were identified; with functional profiles clustering according to pH independent of geography. Differences in gene abundances were found to reflect survival and nutrient acquisition strategies, with organic-rich acidic soils harboring a greater abundance of cation efflux pumps, C and N direct fixation systems and fermentation pathways indicative of anaerobiosis. Conversely, high pH soils possessed more direct transporter-mediated mechanisms for organic C and N substrate acquisition. These findings show that bacterial functional versatility may not be constrained by taxonomy, and we further identify the range of physiological adaptations required to exist in soils of varying nutrient availability and edaphic conditions.

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