Background: Metagenomic sequencing is a well-established tool in the modern biosciences. While it promises unparalleled insights into the genetic content of the biological samples studied, conclusions drawn are at risk from biases inherent to the DNA sequencing methods, including inaccurate abundance estimates as a function of genomic guanine-cytosine (GC) contents. Results: We explored such GC biases across many commonly used platforms in experiments sequencing multiple genomes (with mean GC contents ranging from 28.9% to 62.4%) and metagenomes. GC bias profiles varied among different library preparation protocols and sequencing platforms. We found that our workflows using MiSeq and NextSeq were hindered by major GC biases, with problems becoming increasingly severe outside the 45–65% GC range, leading to a falsely low coverage in GC-rich and especially GC-poor sequences, where genomic windows with 30% GC content had >10-fold less coverage than windows close to 50% GC content. We also showed that GC content correlates tightly with coverage biases. The PacBio and HiSeq platforms also evidenced similar profiles of GC biases to each other, which were distinct from those seen in the MiSeq and NextSeq workflows. The Oxford Nanopore workflow was not afflicted by GC bias. Conclusions: These findings indicate potential sources of difficulty, arising from GC biases, in genome sequencing that could be pre-emptively addressed with methodological optimizations provided that the GC biases inherent to the relevant workflow are understood. Furthermore, it is recommended that a more critical approach be taken in quantitative abundance estimates in metagenomic studies. In the future, metagenomic studies should take steps to account for the effects of GC bias before drawing conclusions, or they should use a demonstrably unbiased workflow.

The importance of the microbial diversity of bioaerosols in relation to occupational exposure and work related health symptoms is not known. The aim of this paper is to gain knowledge on the bacterial and fungal communities in dust causing organic dust toxic syndrome (ODTS) and in reference dust not causing ODTS. Bacterial and fungal communities were described in personal exposure samples from grass seed workers developing ODTS, in dust generated from grass seeds causing ODTS and in dust generated from reference seeds not causing ODTS. Amplicon sequencing of the bacterial 16S rRNA gene and the fungal ITS region, as well as matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) were used for identification of fungi and bacteria in personal exposure samples and in dust samples from grass seeds causing ODTS and in dust from reference grass seeds. Furthermore, activities of enzymes were measured in the same samples. The sequencing data revealed more than 150 bacterial and 25 fungal genera present in each sample. Streptomyces spp., Aspergillus fumigatus and Rhizopus microsporus were dominating in the dust causing ODTS but not in the reference dust. The dustiness in terms of Mucor sp. and R. microsporus were 100-1000 times higher for problematic seeds compared to reference seeds. The bacterial species in the dust causing ODTS included pathogenic species such as Klebsiella pneumonia and Streptomyces pneumonia, and it contained increased concentrations of total protein, serine protease, chitinase, and β-glucosidase. Twenty-three bacterial genera covered more than 50% of the total reads in the personal and problematic seed dust. These 23 genera accounted for less than 7% of the total reads in the reference seed dust. The microbial community of the dust from the problematic seeds showed great similarities to that from the personal air samples from the workers. In conclusion, we have shown for the first time a shift in the microbial community in aerosol samples that caused ODTS compared to the reference samples that did not cause the ODTS. Furthermore, elevated enzyme activities were found in the dust causing ODTS.

Parasitic plants rely on their host to cover their nutritional requirements either for their entire life or a smaller part of it. Depending on the level of parasitism, a proportional reduction on the plastid genome has been found. However, knowledge on gene loss and evolution of the mitogenome of parasitic plants is only available for four hemiparasitic Viscum species (Viscaceae), which lack many of the mitochondrial genes, while the remaining genes exhibit very fast molecular evolution rates. In this study, we include another genus, Phoradendron, from the Viscaceae, as well as 10 other hemiparasitic or holoparasitic taxa from across the phylogeny of the angiosperms to investigate how fast molecular evolution works on their mitogenomes, and the extent of gene loss. Our observations from Viscum were replicated in Phoradendron liga, whereas the remaining parasitic plants in the study have a complete set of the core mitochondrial genes and exhibit moderate or only slightly raised substitution rates compared to most autotrophic taxa, without any statistically significant difference between the different groups (autotrophs, hemiparasites and holoparasites). Additionally, further evidence is provided for the placement of Balanophoraceae within the order Santalales, while the exact placement of Cynomoriaceae still remains elusive. We examine the mitochondrial gene content of 11 hemiparasitic and holoparasitic plants and confirm previous observations in Viscaceae. We show that the remaining parasitic plants do not have significantly higher substitution rates than autotrophic plants in their mitochondrial genes. We provide further evidence for the placement of Balanophoraceae in the Santalales.

Glacier and ice sheet surfaces host diverse communities of microorganisms whose activity (or inactivity) influences biogeochemical cycles and ice melting. Supraglacial microbes endure various environmental extremes including resource scarcity, frequent temperature fluctuations above and below the freezing point of water, and high UV irradiance during summer followed by months of total darkness during winter. One strategy that enables microbial life to persist through environmental extremes is dormancy, which despite being prevalent among microbial communities in natural settings, has not been directly measured and quantified in glacier surface ecosystems. Here, we use a combination of metabarcoding and metatranscriptomic analyses, as well as cell‐specific activity (BONCAT) incubations to assess the diversity and activity of microbial communities from glacial surfaces in Iceland and Greenland. We also present a new ecological model for glacier microorganisms and simulate physiological state‐changes in the glacial microbial community under idealized (i) freezing, (ii) thawing, and (iii) freeze–thaw conditions. We show that a high proportion (>50%) of bacterial cells are translationally active in‐situ on snow and ice surfaces, with Actinomycetota, Pseudomonadota, and Planctomycetota dominating the total and active community compositions, and that glacier microorganisms, even when frozen, could resume translational activity within 24 h after thawing. Our data suggest that glacial microorganisms respond rapidly to dynamic and changing conditions typical of their natural environment. We deduce that the biology and biogeochemistry of glacier surfaces are shaped by processes occurring over short (i.e., daily) timescales, and thus are susceptible to change following the expected alterations to the melt‐regime of glaciers driven by climate change. A better understanding of the activity of microorganisms on glacier surfaces is critical in addressing the growing concern of climate change in Polar regions, as well as for their use as analogues to life in potentially habitable icy worlds.

Two thermophilic trickle bed reactors (TBRs) were packed with different packing densities with polyurethane foam (PUF) and their performance under different retention times were evaluated during ex-situ biogas upgrading process. The results showed that the TBR more tightly packed i.e. containing more layers of PUF achieved higher H₂ utilization efficiency (>99%) and thus, higher methane content (>95%) in the output gas. The tightly packed micro-porous PUF enhanced biofilm immobilization, gas-liquid mass transfer and biomethanation efficiency. Moreover, applying a continuous high-rate nutrient trickling could lead to liquid overflow resulting in formation of non-homogenous biofilm and severe deduction of biomethanation efficiency. High-throughput 16S rRNA gene sequencing revealed that the liquid media were predominated by hydrogenotrophic methanogens. Moreover, members of Peptococcaceae family and uncultured members of Clostridia class were identified as the most abundant species in the biofilm. The proliferation of hydrogenotrophic methanogens together with syntrophic bacteria showed that H₂ addition resulted in altering the microbial community in biogas upgrading process.

Permafrost soils store a substantial part of the global soil carbon and nitrogen. However, global warming causes abrupt erosion and gradual thaw, which make these stocks vulnerable to microbial decomposition into greenhouse gases. Here, we investigated the microbial response to abrupt in situ permafrost thaw. We sequenced the total RNA of a 1 m deep soil core consisting of up to 26 500-year-old permafrost material from an active abrupt erosion site. We analysed the microbial community in the active layer soil, the recently thawed, and the intact permafrost, and found maximum RNA:DNA ratios in recently thawed permafrost indicating a high microbial activity. In thawed permafrost, potentially copiotrophic Burkholderiales and Sphingobacteriales, but also microbiome predators dominated the community. Overall, both thaw-dependent and long-term soil properties significantly correlated with changes in community composition, as did microbiome predator abundance. Bacterial predators were dominated in shallower depths by Myxococcota, while protozoa, especially Cercozoa and Ciliophora, almost tripled in relative abundance in thawed layers. Our findings highlight the ecological importance of a diverse interkingdom and active microbial community highly abundant in abruptly thawing permafrost, as well as predation as potential biological control mechanism.