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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Genetic fingerprinting methods, such as denaturing gradient gel electrophoresis (DGGE), are used in microbial ecology for the analysis of mixed microbial communities but are associated with various problems. In the present study we used a new alternative method: denaturing high-performance liquid chromatography (dHPLC). This method was previously shown to work with samples from water and gut flora but had not yet been applied to complex environmental samples. In contrast to other publications dealing with dHPLC, we used a commonly available HPLC system. Samples from different origins (fermentor sludge, compost, and soil), all ecologically significant, were tested, and the 16S rRNA gene was amplified via PCR. After optimization of the HPLC elution conditions, amplicons of pure cultures and mixed microbial populations could be separated successfully. Systematic differentiation was carried out by a cloning approach, since fraction collection of the peaks did not result in satisfactory fragment separation. dHPLC was evaluated as a tool for microbial community analysis on a genetic level and demonstrated major improvements compared to gel-based fingerprinting methods, such as DGGE, that are commonly used in microbial ecology.The introduction of molecular biological methods in microbiology has changed research in microbial ecology fundamentally. The approach to characterize and classify microbial communities by cultivation methods switched to the genetic level, and the analysis of community structure became possible without any further need of cultivation for systematic analysis. This is especially important since only an estimated 1% of the naturally occurring bacteria have been isolated and characterized thus far (12,15).Genetic fingerprinting techniques, often in combination with a cloning approach, are often used. They provide a pattern or profile of the community diversity based upon the physical separation of unique nucleic acid species, while systematic assignment is often achieved by results from clone libraries. The fingerprinting methods are quite rapid, relatively easy to perform, and permit the simultaneous analysis of multiple samples. This allows comparison of the genetic diversity of microbial communities from different habitats, and in individual communities over time. The most commonly applied methods include denaturing gradient gel electrophoresis (DGGE), temperature gradient gel electrophoresis, single-strand conformation polymorphism, restriction fragment length polymorphism, and terminal restriction fragment length polymorphism. All of these methods provide information on community structure, and DGGE is probably the most frequently used method for this purpose. However, in daily lab use, many problems arise with any of these methods (e.g., the lack of reliable and fast quantification, difficulties in reproducibility, labor-intensive steps, etc.), so alternatives are necessary.In the present study we describe an alternative fingerprinting method, denaturing high-performance liquid chromatography (...

Molecular-microbiological techniques have delivered insight into microbial populations present in anaerobic fermenters, although much information still remains to be elucidated. In this study, the ability of denaturing gradient gel electrophoresis (DGGE) to throw light on microbial community composition was investigated and latter data were compared with the gas production of a 750,000l anaerobic biogas fermenter. During 1 year, samples were taken from two different sites of the reactor and additionally from the substrate material. After DNA extraction and PCR with archaeal and bacterial primers, PCR products were run on denaturing gradient gels to compare band patterns. Using gel-imaging software (GelComparII), two major clusters could be identified. Dominant bands were excised from the gels, reamplified and sequenced. Most sequences were closely related to Lactobacilli and yet uncultured microorganisms. DNA content of all samples was significantly correlated with the gas production measured online. We concluded that PCR and subsequent DGGE are useful to monitor community shifts in anaerobic fermenter sludge. However, as these changes are not readily detectable via DGGE-pattern analysis, alternative factors influencing the fermenter functioning should be found and investigated. So far DNA-content measurement seems to be a good parameter to quickly determine anaerobic fermenter condition.

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