Although of limited metabolic diversity, methanogenic archaea or methanogens possess great phylogenetic and ecological diversity. Only three types of methanogenic pathways are known: CO(2)-reduction, methyl-group reduction, and the aceticlastic reaction. Cultured methanogens are grouped into five orders based upon their phylogeny and phenotypic properties. In addition, uncultured methanogens that may represent new orders are present in many environments. The ecology of methanogens highlights their complex interactions with other anaerobes and the physical and chemical factors controlling their function.

Metabolic, phylogenetic, and ecological diversity of the methanogenic archaea.

Anaerobic conversion of organic wastes and biomass to methane is an important bioenergy strategy, which depends on poorly understood mechanisms of interspecies electron transfer to methanogenic microorganisms. Metatranscriptomic analysis of methanogenic aggregates from a brewery wastewater digester, coupled with fluorescence in situ hybridization with specific 16S rRNA probes, revealed that Methanosaeta species were the most abundant and metabolically active methanogens. Methanogens known to reduce carbon dioxide with H2 or formate as the electron donor were rare. Although Methanosaeta have previously been thought to be restricted to acetate as a substrate for methane production, Methanosaeta in the aggregates had a complete complement of genes for the enzymes necessary for the reduction of carbon to methane, and transcript abundance for these genes was high. Furthermore, Geobacter species, the most abundant bacteria in the aggregates, highly expressed genes for ethanol metabolism and for extracellular electron transfer via electrically conductive pili, suggesting that Geobacter and Methanosaeta species were exchanging electrons via direct interspecies electron transfer (DIET). This possibility was further investigated in defined co-cultures of Geobacter metallireducens and Methanosaeta harundinacea which stoichiometrically converted ethanol to methane. Transcriptomic, radiotracer, and genetic analysis demonstrated that M. harundinacea accepted electrons via DIET for the reduction of carbon dioxide to methane. The discovery that Methanosaeta species, which are abundant in a wide diversity of methanogenic environments, are capable of DIET has important implications not only for the functioning of anaerobic digesters, but also for global methane production.

https://bio.as.uky.edu/sites/default/files/Rotaru%20et%20al.%202014%20EES%20copy.pdf

A new model for electron flow during anaerobic digestion: direct interspecies electron transfer to Methanosaeta for the reduction of carbon dioxide to methane

Many bacterial species have been found to exist in a viable but non-culturable (VBNC) state since its discovery in 1982. VBNC cells are characterized by a loss of culturability on routine agar, which impairs their detection by conventional plate count techniques. This leads to an underestimation of total viable cells in environmental or clinical samples, and thus poses a risk to public health. In this review, we present recent findings on the VBNC state of human bacterial pathogens. The characteristics of VBNC cells, including the similarities and differences to viable, culturable cells and dead cells, and different detection methods are discussed. Exposure to various stresses can induce the VBNC state, and VBNC cells may be resuscitated back to culturable cells under suitable stimuli. The conditions that trigger the induction of the VBNC state and resuscitation from it are summarized and the mechanisms underlying these two processes are discussed. Last but not least, the significance of VBNC cells and their potential influence on human health are also reviewed.

/pdf/the-importance-of-the-viable-but-non-culturable-state-in-29lc2uaevt.pdf

The importance of the viable but non-culturable state in human bacterial pathogens.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1574-6941.2008.00629.x

Advantages and limitations of quantitative PCR (Q-PCR)-based approaches in microbial ecology.

Methane (CH4) is an important greenhouse gas because it has 25 times the global warming potential of carbon dioxide (CO2) by mass over a century. Recent calculations suggest that atmospheric CH4 emissions have been responsible for approximately 20% of Earth's warming since pre-industrial times. Understanding how CH4 emissions from ecosystems will respond to expected increases in global temperature is therefore fundamental to predicting whether the carbon cycle will mitigate or accelerate climate change. Methanogenesis is the terminal step in the remineralization of organic matter and is carried out by strictly anaerobic Archaea. Like most other forms of metabolism, methanogenesis is temperature-dependent. However, it is not yet known how this physiological response combines with other biotic processes (for example, methanotrophy, substrate supply, microbial community composition) and abiotic processes (for example, water-table depth) to determine the temperature dependence of ecosystem-level CH4 emissions. It is also not known whether CH4 emissions at the ecosystem level have a fundamentally different temperature dependence than other key fluxes in the carbon cycle, such as photosynthesis and respiration. Here we use meta-analyses to show that seasonal variations in CH4 emissions from a wide range of ecosystems exhibit an average temperature dependence similar to that of CH4 production derived from pure cultures of methanogens and anaerobic microbial communities. This average temperature dependence (0.96 electron volts (eV)), which corresponds to a 57-fold increase between 0 and 30°C, is considerably higher than previously observed for respiration (approximately 0.65 eV) and photosynthesis (approximately 0.3 eV). As a result, we show that both the emission of CH4 and the ratio of CH4 to CO2 emissions increase markedly with seasonal increases in temperature. Our findings suggest that global warming may have a large impact on the relative contributions of CO2 and CH4 to total greenhouse gas emissions from aquatic ecosystems, terrestrial wetlands and rice paddies.

/pdf/methane-fluxes-show-consistent-temperature-dependence-across-3mb51rrrfi.pdf

Methane fluxes show consistent temperature dependence across microbial to ecosystem scales

Temperature is an important factor controlling CH4production in anoxic rice soils. Soil slurries, prepared from Italian rice field soil, were incubated anaerobically in the dark at six temperatures of between 10 to 37°C or in a temperature gradient block covering the same temperature range at intervals of 1°C. Methane production reached quasi-steady state after 60 to 90 days. Steady-state CH4 production rates increased with temperature, with an apparent activation energy of 61 kJ mol−1. Steady-state partial pressures of the methanogenic precursor H2 also increased with increasing temperature from

Effect of temperature on carbon and electron flow and on the archaeal community in methanogenic rice field soil.

Quantitative PCR (Q-PCR) is a fast and efficient tool to quantify target genes. In eukaryotic cells, quantitative reverse transcription-PCR (Q-RT-PCR) is also used to quantify gene expression, with stably expressed housekeeping genes as standards. In bacteria, such stable expression of housekeeping genes does not occur, and the use of DNA standards leads to a broad underestimation. Therefore, an accurate quantification of RNA is feasible only by using appropriate RNA standards. We established and validated a Q-PCR method which enables the quantification of not only the number of copies of target genes (i.e., the number of bacterial cells) but also the number of RNA copies. The genes coding for InvA and the 16S rRNA of Salmonella enterica serovar Typhimurium were selected for the evaluation of the method. As DNA standards, amplified fragments of the target genes were used, whereas the same DNA standards were transcribed in vitro for the development of appropriate RNA standards. Salmonella cultures and environmental water samples inoculated with bacteria were then employed for the final testing. Both experimental approaches led to a sensitive, accurate, and reproducible quantification of the selected target genes and RNA molecules by Q-PCR and Q-RT-PCR. It is the first time that RNA standards have been successfully used for a precise quantification of the number of RNA molecules in prokaryotes. This demonstrates the potential of this approach for determining the presence and metabolic activity of pathogenic bacteria in environmental samples.

Establishment of a Real-Time PCR-Based Approach for Accurate Quantification of Bacterial RNA Targets in Water, Using Salmonella as a Model Organism

Temporal change of 13C-isotope signatures and methanogenic pathways in rice field soil incubated anoxically at different temperatures

Summary

We performed a comparative analysis of the Vibrio cholerae strain El Tor 3083 entering the viable but non-culturable (VBNC) state and starvation after incubation in artificial seawater (ASW) at 4 and 15°C respectively. To this end, we determined bacterial culturability and membrane integrity, as well as the cellular levels of 16S rRNA and mRNA for the tuf, rpoS and relA genes, which were assessed by real-time quantitative reverse transcription polymerase chain reaction (Q-RT-PCR). Bacterial cells entering the VBNC state showed a 154, 5.1 × 103, 24- and 23-fold reduction in the number of copies of 16S rRNA and mRNA for tuf, rpoS and relA, in comparison to exponentially growing cells. The differences were less striking between cells in the VBNC and starvation states. The mRNA for relA was selectively increased in VBNC cells (3.2-folds), whereas a 3.9-fold reduction was observed for 16S rRNA. The obtained results confirmed that key activities of the cellular metabolism (i.e. tuf representing protein synthesis, and relA or rpoS stress response) were still detected in bacteria entering the VBNC state and starvation. These data suggest that the new Q-RT-PCR methodology, based on the selected RNA targets, could be successfully exploited for the identification (rRNA) of V. cholerae and assessment of its metabolic activity (tuf, rpoS, relA mRNA) in environmental samples.

/pdf/quantitative-reverse-transcription-polymerase-chain-reaction-4ryep2l5e3.pdf

Quantitative reverse transcription polymerase chain reaction analysis of Vibrio cholerae cells entering the viable but non-culturable state and starvation in response to cold shock

Temperature is an important factor controlling CH 4 production in rice field soils. However, it is unknown which step in the methanogenic degradation of organic matter is the limiting one that is controlled by temperature. Soil slurries prepared from Italian rice field soil were anaerobically incubated in the dark at six different temperatures between 10 and 37 °C until quasi-steady state was reached. Then, the potential and actual rates of polysaccharide hydrolysis and of CH 4 production from different immediate (acetate, H 2 ) and distal (glucose, propionate) methanogenic substrates were determined. Potential activities of exo -glucanase and glucosidase were always higher than the actual rates of polysaccharide hydrolysis indicating that the availability of the polysaccharide substrate was limiting at all temperatures. The actual rates of CH 4 production were always lower than those predicted from glucose release during polysaccharide hydrolysis indicating that a substantial amount of the released glucose was assimilated into microbial biomass. Addition of the different methanogenic substrates stimulated CH 4 production at all temperatures >10 °C, but only at >20 °C to values higher than rates of polysaccharide hydrolysis. Under steady state conditions, however, hydrolysis of organic polymers was the rate-limiting step at all temperatures >10 °C.

Axel Fey

Papers

Effect of temperature on carbon and electron flow and on the archaeal community in methanogenic rice field soil.

Establishment of a Real-Time PCR-Based Approach for Accurate Quantification of Bacterial RNA Targets in Water, Using Salmonella as a Model Organism

Temporal change of 13C-isotope signatures and methanogenic pathways in rice field soil incubated anoxically at different temperatures

Quantitative reverse transcription polymerase chain reaction analysis of Vibrio cholerae cells entering the viable but non-culturable state and starvation in response to cold shock

Effect of temperature on the rate limiting step in the methanogenic degradation pathway in rice field soil