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 ...

SARS-CoV-2 surveillance by wastewater-based epidemiology is poised to provide a complementary approach to sequencing individual cases. However, robust quantification of variants and de novo detection of emerging variants remains challenging for existing strategies. We deep sequenced 3,413 wastewater samples representing 94 municipal catchments, covering >59% of the population of Austria, from December 2020 to February 2022. Our system of variant quantification in sewage pipeline designed for robustness (termed VaQuERo) enabled us to deduce the spatiotemporal abundance of predefined variants from complex wastewater samples. These results were validated against epidemiological records of >311,000 individual cases. Furthermore, we describe elevated viral genetic diversity during the Delta variant period, provide a framework to predict emerging variants and measure the reproductive advantage of variants of concern by calculating variant-specific reproduction numbers from wastewater. Together, this study demonstrates the power of national-scale WBE to support public health and promises particular value for countries without extensive individual monitoring.

With regard to social and environmental sustainability, second-generation biofuel and biogas production from lignocellulosic material provides considerable potential, since lignocellulose represents an inexhaustible, ubiquitous natural resource, and is therefore one important step towards independence from fossil fuel combustion. However, the highly heterogeneous structure and recalcitrant nature of lignocellulose restricts its commercial utilization in biogas plants. Improvements therefore rely on effective pretreatment methods to overcome structural impediments, thus facilitating the accessibility and digestibility of (ligno)cellulosic substrates during anaerobic digestion. While chemical and physical pretreatment strategies exhibit inherent drawbacks including the formation of inhibitory products, biological pretreatment is increasingly being advocated as an environmentally friendly process with low energy input, low disposal costs, and milder operating conditions. Nevertheless, the promising potential of biological pretreatment techniques is not yet fully exploited. Hence, we intended to provide a detailed insight into currently applied pretreatment techniques, with a special focus on biological ones for downstream processing of lignocellulosic biomass in anaerobic digestion.

BackgroundSubstrate spectra for anaerobic digestion have been broadened in the past decade, inter alia, due to the application of different pretreatment strategies and now include materials rich in lignocellulose, protein, and/or fat. The application of these substrates, however, also entails risks regarding the formation of undesired by-products, among which phenolic compounds are known to accumulate under unfavorable digestion conditions.MethodsDifferent states of overload were simulated in batch experiments while reviewing the generation of phenyl acids out of different lab-use substrates in order to evaluate the impact on biogas and methane production as well as some additional process performance parameters under defined laboratory conditions. Investigations were conducted under both mesophilic and thermophilic conditions.ResultsIt could be shown that the tested input materials led to the formation of phenyl acids in a substrate-dependent manner with the formation itself being less temperature driven. Once formed, the formation of phenyl acids turned out to be a reversible process.ConclusionsAlthough a mandatory negative impact of phenyl acids per se on the anaerobic digestion process in general and the methanogenesis process in particular could not be proven, phenyl acids, however, seem to play an important role in the microbial response to overloaded biogas systems.Electronic supplementary materialThe online version of this article (10.1186/s13068-019-1370-6) contains supplementary material, which is available to authorized users.

HighlightsWe evaluated different soil DNA extraction procedures and disintegration strategies.All tests were conducted using a reference soil.We tested repeated DNA extractions (up to 10 times).A method (EMA; PMA) for the discrimination of cells and free DNA was tested.DNA yield is affected by extraction procedure, microbial diversity merely.