Influence of different sample preparation techniques on imaging viruses and virus-like particles by scanning electron and scanning transmission electron microscopes.

Scanning electron microscopy (SEM) and scanning transmission electron microscopy (STEM) were applied in many laboratories to visualize and image viruses and virus-like particles (VLPs). Two bacteriophages, P1 and Φ6, were chosen as model microorganisms known for their distinct structure, and viruses obtained from biofilms associated with modern travertines (Terme di Saturnia, Italy; Karahayit "Kizilsu" and Pamukkale, Turkey) were also investigated. Three protocols, (1) full, (2) simplified, and (3) all at once were developed and tested for sample preparation and imaging. The full procedure enabled the observation of P1 bacteriophages, whereas the simplified protocol, successful in visualizing Φ6, did not yield satisfactory results for P1. The preservation state of the latter appeared to be compromised and led to less informative images in SEM and STEM. Viruses in biofilms exhibited various levels of mineralization and aggregation, complicating their characterization. In the all at once procedure, although effective in preserving bacteriophage tails, excessive coating and thickening of samples with heavy chemical reagents led to a reduction in overall image quality. Despite a final washing step, some residues of chemical reagents (OsO4 and uranyl acetate) remained, impacting the clarity of the images. Finally, the results obtained emphasize the critical role of sample preparation and imaging techniques in effectively visualizing and characterizing viruses and VLPs. The choice of analytical procedure significantly influences the resolution and preservation state of the observed bacteriophages and VLPs. It is suggested that the appropriate imaging technique is carefully selected based on the specific objectives of the project and the nature of the samples being investigated to obtain the best images of the viruses.

Viruses participate in the organomineralization of travertines.

Travertines, which precipitate from high temperature water saturated with calcium carbonate, are generally considered to be dominated by physico-chemical and microbial precipitates. Here, as an additional influence on organomineral formation, metagenomic data and microscopic analyses clearly demonstrate that highly diverse viral, bacterial and archaeal communities occur in the biofilms associated with several modern classic travertine sites in Europe and Asia, along with virus-like particles. Metagenomic analysis reveals that bacteriophages (bacterial viruses) containing icosahedral capsids and belonging to the Siphoviridae, Myoviridae and Podoviridae families are the most abundant of all viral strains, although the bacteriophage distribution does vary across the sampling sites. Icosahedral shapes of capsids are also the most frequently observed under the microscope, occurring as non-mineralized through to mineralized viruses and virus-like particles. Viruses are initially mineralized by Ca-Si amorphous precipitates with subordinate Mg and Al contents; these then alter to nanospheroids composed of Ca carbonate with minor silicate 80-300 nm in diameter. Understanding the roles of bacteriophages in modern carbonate-saturated settings and related organomineralization processes is critical for their broader inclusion in the geological record and ecosystem models.

Microbial Involvement in Carbon Transformation via CH4 and CO2 in Saline Sedimentary Pool.

Methane and carbon dioxide are one of the most important greenhouse gases and significant components of the carbon cycle. Biogeochemical methane transformation may occur even in the extreme conditions of deep subsurface ecosystems. This study presents methane-related biological processes in saline sediments of the Miocene Wieliczka Formation, Poland. Rock samples (W2, W3, and W4) differed in lithology (clayey salt with veins of fibrous salt and lenses of gypsum and anhydrite; siltstone and sandstone; siltstone with veins of fibrous salt and lenses of anhydrite) and the accompanying salt type (spiza salts or green salt). Microbial communities present in the Miocene strata were studied using activity measurements and high throughput sequencing. Biological activity (i.e., carbon dioxide and methane production or methane oxidation) occurred in all of the studied clayey salt and siltstone samples but mainly under water-saturated conditions. Microcosm studies performed at elevated moisture created more convenient conditions for the activity of both methanogenic and methanotrophic microorganisms than the intact sediments. This points to the fact that water activity is an important factor regulating microbial activity in saline subsurface sediments. Generally, respiration was higher in anaerobic conditions and ranged from 36 ± 2 (W2200%t.w.c) to 48 ± 4 (W3200%t.w.c) nmol CO2 gdw-1 day-1. Methanogenic activity was the highest in siltstone and sandstone (W3, 0.025 ± 0.018 nmol CH4 gdw-1 day-1), while aerobic methanotrophic activity was the highest in siltstone with salt and anhydrite (W4, 220 ± 66 nmol CH4 gdw-1 day-1). The relative abundance of CH4-utilizing microorganisms (Methylomicrobium, Methylomonas, Methylocystis) constituted 0.7-3.6% of all taxa. Methanogens were represented by Methanobacterium (0.01-0.5%). The methane-related microbes were accompanied by a significant number of unclassified microorganisms (3-64%) and those of the Bacillus genus (4.5-91%). The stable isotope composition of the CO2 and CH4 trapped in the sediments suggests that methane oxidation could have influenced δ13CCH4, especially in W3 and W4.

A survey of greenhouse gases production in central European lignites.

Due to changes in the energy market, it is projected that lignite excavation will be reduced in the near future. Cessation of exploitation is associated with restitution of natural water conditions and flooding of the resources left in the mines. Flooded lignite mines are a potential source of greenhouse gases (GHG) (CH4, CO2 and N2O), which should be monitored due to growing environmental concerns. Here, we aim to recognize GHG release from the lignites collected from the main deposits of Poland, Slovenia and Serbia. GHG production was studied along with a range of physical and chemical parameters that are crucial for microbial growth and activity. The microcosm experiments showed that the main gas emitted from the lignites was carbon dioxide. Daily CO2 production was highly variable. The highest values were recorded for detroxylitic lignite collected from the Konin deposit (402.05 nmol CO2 g-1 day-1) while the lowest were for the Kolubara xylitic lignite (19.64 nmol CO2 g-1 day-1). Methane production was much lower and ranged from nearly zero to 66.75 nmol g dry mass-1 d-1. Nitrous oxide production was not detected. It was found that CO2 production, being a general measure of microbial activity, was positively affected by NO3- concentration and redox potential. With respect to methane formation, the lower atmospheric oxygen exposure of the sample from the Velenje underground mine compared to the samples from the opencast mines has been identified as a possible cause of the high methane production. The overall global warming potential (GWP) of the gases released by xylitic lignite was lowest among the samples. Preferential extraction of the detritic lignites is suggested as a means to reduce GHG emissions from the abandoned lignite mines.