We studied the effects of applying different composts (urban organic waste, green waste, manure and sewage sludge), mineral fertilizer and compost plus mineral fertilizer on chemical, biological and soil microbiological parameters over a 12-year period. The organic C and total N levels in soils were increased by all compost and compost + N treatments. Microbial biomass C was significantly (P £ 0.05) increased for some compost treatments. In addition, basal respiration and the metabolic quotient (qCO 2 ) were significantly higher in all soils that had received sewage sludge compost. The Shannon diversity index (H), based on community level physiological profiling, showed a higher consumption of carbon sources in soils treated with compost and compost + N compared with the control. The utilization of different guilds of carbon sources varied amongst the treatments (compost, compost + N or mineral fertilizer). Cluster analysis of polymerase chain reaction-denaturing gradient gel electrophoresis patterns showed two major clusters, the first containing the mineral fertilization and compost treatments, and the second, the composts + N treatments. No differences in bacterial community structure could be determined between the different types of compost. However, the results suggest that long-term compost treatments do have effects on the soil biota. The results indicate that the effects on the qCO 2 may be due to shifts in community composition. In this study, it was not possible to distinguish with certainty between the effects of different composts except for compost derived from sewage sludge.

Recently, new DNA extraction techniques (using ethidium monoazide and propidium monoazide) have been developed to discriminate between alive and dead bacterial cells. Nevertheless, for complex environmental samples, no data are available yet. In the present study, these new methods were applied to anaerobicfermentor sludge and the results were compared to a conventional microbiological approach.For pathogen risk assessment and hygienic safety control in anaerobic digesters, various culture-based microbiological methods are in use. However, with the application of classical methods, a number of problems arise: long cultivation times for some microorganisms, the complexity of anaerobic cultivation, and timeconsuming lab work (enrichment of selected organisms, selective cultivation, and subsequent systematic differentiation). Alternatively, molecular tools could be used, but fast and easy methods, such as PCR amplification after conventional DNA extraction, do not always guarantee the amplification of viable cells' DNA only (6), which might result in false-positive data (9). On the other hand, RNA-based approaches, which would target the active part of a microbial community, thus enabling discrimination between living and dead cells, encounter problems with the high RNA decay rates after the loss of cell viability (1) and are also expensive and laborious.A new DNA extraction technique including an additional step to remove free, extracellular DNA and DNA of dead bacterial cells by using light-activated ethidium monoazide (EMA) or propidium monoazide (PMA) was described previously, noting the possibility of a selective suppression of DNA detection in dead cells (10,11,15). To our knowledge, these extraction procedures were tested successfully with a simple matrix (12), whereas an evaluation of environmental matrices, such as the sludge of an anaerobic digestion plant, has not yet been performed.The aim of this work was to test the suitability of EMA and PMA for the extraction of free DNA and DNA originating from dead cells in an environmental matrix. The extracted DNA was subsequently amplified via real-time PCR (quantitative PCR [qPCR]) using specific primers for selected pathogenic microorganisms (Clostridium perfringens, Listeria monocytogenes, and Salmonella enterica), and the results were compared to classical cultivation-based agar plating data.The following organisms, selected after an Austrian standard guideline (14), and an anaerobic spore-forming microorganism, were used after microscopic verification and selective plate counting: Clostridium perfringens (DSM 11780; German Collection of Microorganisms and Cell Cultures, http://www .dsmz.de), Listeria monocytogenes (DSM 15675), and Salmonella enterica subsp. enterica serovar Senftenberg (DSM 10062). Pure cultures of L. monocytogenes and C. perfringens were grown in DSM medium 92 (30.0 g Trypticase soy broth, 3.0 g yeast extract, 1,000 ml distilled water, pH 7.0), and pure cultures of S. enterica were grown in DSM medium 220 (15.0 g peptone from casein, 5.0 ...

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Carbon (C) uptake by terrestrial ecosystems represents an important option for partially mitigating anthropogenic CO2 emissions. Short-term atmospheric elevated CO2 exposure has been shown to create major shifts in C flow routes and diversity of the active soil-borne microbial community. Long-term increases in CO2 have been hypothesized to have subtle effects due to the potential adaptation of soil microorganism to the increased flow of organic C. Here, we studied the effects of prolonged elevated atmospheric CO2 exposure on microbial C flow and microbial communities in the rhizosphere. Carex arenaria (a nonmycorrhizal plant species) and Festuca rubra (a mycorrhizal plant species) were grown at defined atmospheric conditions differing in CO2 concentration (350 and 700 ppm) for 3 years. During this period, C flow was assessed repeatedly (after 6 months, 1, 2, and 3 years) by (13) C pulse-chase experiments, and label was tracked through the rhizosphere bacterial, general fungal, and arbuscular mycorrhizal fungal (AMF) communities. Fatty acid biomarker analyses and RNA-stable isotope probing (RNA-SIP), in combination with real-time PCR and PCR-DGGE, were used to examine microbial community dynamics and abundance. Throughout the experiment the influence of elevated CO2 was highly plant dependent, with the mycorrhizal plant exerting a greater influence on both bacterial and fungal communities. Biomarker data confirmed that rhizodeposited C was first processed by AMF and subsequently transferred to bacterial and fungal communities in the rhizosphere soil. Over the course of 3 years, elevated CO2 caused a continuous increase in the (13) C enrichment retained in AMF and an increasing delay in the transfer of C to the bacterial community. These results show that, not only do elevated atmospheric CO2 conditions induce changes in rhizosphere C flow and dynamics but also continue to develop over multiple seasons, thereby affecting terrestrial ecosystems C utilization processes.

A microarray spotted with 369 different 16S rRNA gene probes specific to microorganisms involved in the degradation process of organic waste during composting was developed. The microarray was tested with pure cultures, and of the 30,258 individual probe-target hybridization reactions performed, there were only 188 false positive (0.62%) and 22 false negative signals (0.07%). Labeled target DNA was prepared by polymerase chain reaction amplification of 16S rRNA genes using a Cy5-labeled universal bacterial forward primer and a universal reverse primer. The COMPOCHIP microarray was applied to three different compost types (green compost, manure mix compost, and anaerobic digestate compost) of different maturity (2, 8, and 16 weeks), and differences in the microorganisms in the three compost types and maturity stages were observed. Multivariate analysis showed that the bacterial composition of the three composts was different at the beginning of the composting process and became more similar upon maturation. Certain probes (targeting Sphingobacterium, Actinomyces, Xylella/Xanthomonas/ Stenotrophomonas, Microbacterium, Verrucomicrobia, Planctomycetes, Low G + C and Alphaproteobacteria) were more influential in discriminating between different composts. Results from denaturing gradient gel electrophoresis supported those of microarray analysis. This study showed that the COMPOCHIP array is a suitable tool to study bacterial communities in composts.

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