The use of extracellular DNA as a proxy for specific microbial activity.

The ubiquity and relevance of extracellular DNA (exDNA) are well-known and increasingly gaining importance in many fields of application such as medicine and environmental microbiology. Although sources and types of exDNA are manifold, ratios of specific DNA-molecules inside and outside of living cells can give reliable information about the activity of entire systems and of specific microbial groups or species. Here, we introduce a method to discriminate between internal (iDNA), as well as bound and free exDNA, and evaluate various DNA fractions and related ratios (ex:iDNA) regarding their applicability to be used as a fast, convenient, and reliable alternative to more tedious RNA-based activity measurements. In order to deal with microbial consortia that can be regulated regarding their activity, we tested and evaluated the proposed method in comparison to sophisticated dehydrogenase- and RNA-based activity measurements with two anaerobic microbial consortia (anaerobic fungi and syntrophic archaea and a microbial rumen consortium) and three levels of resolution (overall activity, total bacteria, methanogenic archaea). Furthermore, we introduce a 28S rRNA gene-specific primer set and qPCR protocol, targeting anaerobic fungi (Neocallimastigomycota). Our findings show that the amount of actively released free exDNA (fDNA) strongly correlates with different activity measurements and is thus suggested to serve as a proxy for microbial activity.

New aspects and strategies for methane mitigation from ruminants.

The growing demand for sustainable animal production is compelling researchers to explore the potential approaches to reduce emissions of greenhouse gases from livestock that are mainly produced by enteric fermentation. Some potential solutions, for instance, the use of chemical inhibitors to reduce methanogenesis, are not feasible in routine use due to their toxicity to ruminants, inhibition of efficient rumen function or other transitory effects. Strategies, such as use of plant secondary metabolites and dietary manipulations have emerged to reduce the methane emission, but these still require extensive research before these can be recommended and deployed in the livestock industry sector. Furthermore, immunization vaccines for methanogens and phages are also under investigation for mitigation of enteric methanogenesis. The increasing knowledge of methanogenic diversity in rumen, DNA sequencing technologies and bioinformatics have paved the way for chemogenomic strategies by targeting methane producers. Chemogenomics will help in finding target enzymes and proteins, which will further assist in the screening of natural as well chemical inhibitors. The construction of a methanogenic gene catalogue through these approaches is an attainable objective. This will lead to understand the microbiome function, its relation with the host and feeds, and therefore, will form the basis of practically viable and eco-friendly methane mitigation approaches, while improving the ruminant productivity.

Enumeration of methanogens with a focus on fluorescence in situ hybridization.

Methanogens, the members of domain Archaea are potent contributors in global warming. Being confined to the strict anaerobic environment, their direct cultivation as pure culture is quite difficult. Therefore, a range of culture-independent methods have been developed to investigate their numbers, substrate uptake patterns, and identification in complex microbial communities. Unlike other approaches, fluorescence in situ hybridization (FISH) is not only used for faster quantification and accurate identification but also to reveal the physiological properties and spatiotemporal dynamics of methanogens in their natural environment. Aside from the methodological aspects and application of FISH, this review also focuses on culture-dependent and -independent techniques employed in enumerating methanogens along with associated problems. In addition, the combination of FISH with micro-autoradiography that could also be an important tool in investigating the activities of methanogens is also discussed.

Anaerobic Fungi and Their Potential for Biogas Production.

Plant biomass is the largest reservoir of environmentally friendly renewable energy on earth. However, the complex and recalcitrant structure of these lignocellulose-rich substrates is a severe limitation for biogas production. Microbial pro-ventricular anaerobic digestion of ruminants can serve as a model for improvement of converting lignocellulosic biomass into energy. Anaerobic fungi are key players in the digestive system of various animals, they produce a plethora of plant carbohydrate hydrolysing enzymes. Combined with the invasive growth of their rhizoid system their contribution to cell wall polysaccharide decomposition may greatly exceed that of bacteria. The cellulolytic arsenal of anaerobic fungi consists of both secreted enzymes, as well as extracellular multi-enzyme complexes called cellulosomes. These complexes are extremely active, can degrade both amorphous and crystalline cellulose and are probably the main reason of cellulolytic efficiency of anaerobic fungi. The synergistic use of mechanical and enzymatic degradation makes anaerobic fungi promising candidates to improve biogas production from recalcitrant biomass. This chapter presents an overview about their biology and their potential for implementation in the biogas process.

Coupled cryoconite ecosystem structure-function relationships are revealed by comparing bacterial communities in alpine and Arctic glaciers.

Cryoconite holes are known as foci of microbial diversity and activity on polar glacier surfaces, but are virtually unexplored microbial habitats in alpine regions. In addition, whether cryoconite community structure reflects ecosystem functionality is poorly understood. Terminal restriction fragment length polymorphism and Fourier transform infrared metabolite fingerprinting of cryoconite from glaciers in Austria, Greenland and Svalbard demonstrated cryoconite bacterial communities are closely correlated with cognate metabolite fingerprints. The influence of bacterial-associated fatty acids and polysaccharides was inferred, underlining the importance of bacterial community structure in the properties of cryoconite. Thus, combined application of T-RFLP and FT-IR metabolite fingerprinting promises high throughput, and hence, rapid assessment of community structure-function relationships. Pyrosequencing revealed Proteobacteria were particularly abundant, with Cyanobacteria likely acting as ecosystem engineers in both alpine and Arctic cryoconite communities. However, despite these generalities, significant differences in bacterial community structures, compositions and metabolomes are found between alpine and Arctic cryoconite habitats, reflecting the impact of local and regional conditions on the challenges of thriving in glacial ecosystems.