Validating digital polymerase chain reaction for 16S rRNA gene amplification from low biomass environmental samples.

Digital polymerase chain reaction (dPCR) is a DNA quantification technology that offers absolute quantification of DNA templates. In this study, we optimized and validated a chip-based dPCR EvaGreen assay with commonly used 16S rRNA gene primer pairs and compared its performance to quantitative real-time PCR (qPCR). We compared measurements of low amounts of template DNA using a newly designed synthetic DNA standard to assess precision, accuracy, and sensitivity.

Effect of temperature on microbial communities in bentonite for use in engineered barrier systems.

Bentonite is an important component of deep geological repository (DGR) designs, where it will serve as a buffer between used fuel containers (UFCs) and subsurface rock walls of the repository. The potential for microbial activity in bentonite is being studied to understand the influence of microbial metabolisms (e.g.

Interface-associated microbial growth in anoxic pressure vessels containing compacted gapfill bentonite.

Aims: Deep geological repositories (DGR) for belowground storage of used nuclear fuel rely on highly compacted bentonite to limit growth of microorganisms, including those that can contribute to microbiologically influenced corrosion, like sulfate-reducing bacteria (SRB). This study examined how a range of bentonite dry densities (1.25-1.

Low water activity limits bentonite-associated microbial growth.

Aims: This research investigated the impact of water activity on microbial abundance estimates from bentonite under conditions relevant to a deep geological repository for used nuclear fuel. Because previous research tested saturation of bentonite within pressurized vessels, the goal of this study was to assess how water activity alone, without pressure, prevents increases in microbial abundance estimates from bentonite samples.

Methods And Results: Small-scale microcosms of bentonite were hydrated to low, medium, or high water activities, with Type I water, reference groundwater, or bacterial growth medium, then incubated under oxic or anoxic conditions.

Comparative proteomics of biofilm development in discovers a distinct family of Ca-dependent adhesins.

Unlabelled: The marine bacterium, , is a useful model for studying biofilm development due to its ability to colonize and form biofilms on a variety of marine and eukaryotic host-associated surfaces. However, the pathways responsible for biofilm formation are not fully understood, in part due to a lack of functional information for a large proportion of its proteome. We used comparative shotgun proteomics to explore biofilm development from the planktonic phase throughout early, middle, and late biofilm stages.

Minimal changes in microbial abundances and diversity over 7 years of emplacement for modules of compacted bentonite exposed to natural groundwater.

The multi-national Materials Corrosion Test (MaCoTe), being conducted at the Grimsel Test Site in Switzerland, assesses the stability of bentonite as it may be utilized within deep geological repositories (DGRs), which are proposed for the safe, long-term disposal of used nuclear fuel. This experiment provides an opportunity for long-term assessments of changes in microbial communities associated with compacted subsurface bentonite samples exposed to a natural groundwater. Leveraging samples from MaCoTe, herein, we report temporal data for the abundance and community composition of microorganisms associated with compacted bentonite samples emplaced over 7 years under subsurface conditions.

Characterizing biofilm thickness, density, and microbial community composition in a full-scale hybrid membrane aerated biofilm reactor.

This study examined biofilm thickness, density, and microbial composition in a full-scale MABR treating municipal wastewater, focusing on their spatial and operational variability. The MABR cassette arrangement created a thickness gradient, with biofilms in the front cassettes more than twice as thick as those at the back. Lower scouring intensity due to reduced airflow resulted in thicker biofilms.

Deep terrestrial indigenous microbial community dominated by Frackibacter.

Characterizing deep subsurface microbial communities informs our understanding of Earth's biogeochemistry as well as the search for life beyond the Earth. Here we characterized microbial communities within the Kidd Creek Observatory subsurface fracture water system with mean residence times of hundreds of millions to over one billion years. 16S rRNA analysis revealed that biosamplers well isolated from the mine environment were dominated by a putatively anaerobic and halophilic bacterial species from the family, Frackibacter.

Comammox among dominant ammonia oxidizers within aquarium biofilter microbial communities.

Unlabelled: Nitrification by aquarium biofilters transforms ammonia waste (NH/NH) to less toxic nitrate (NO) via nitrite (NO). Prior to the discovery of complete ammonia-oxidizing ("comammox" or CMX) , previous research revealed that ammonia-oxidizing archaea (AOA) dominated over ammonia-oxidizing bacteria (AOB) in freshwater aquarium biofilters. Here, we profiled aquarium biofilter microbial communities and quantified the abundance of all three known ammonia oxidizers using 16S rRNA gene sequencing and quantitative PCR (qPCR), respectively.

Microbial ecology of the deep terrestrial subsurface.

The terrestrial subsurface hosts microbial communities that, collectively, are predicted to comprise as many microbial cells as global surface soils. Although initially thought to be associated with deposited organic matter, deep subsurface microbial communities are supported by chemolithoautotrophic primary production, with hydrogen serving as an important source of electrons. Despite recent progress, relatively little is known about the deep terrestrial subsurface compared to more commonly studied environments.

Microbial communities change along the 300 km length of the Grand River for extreme high- and low-flow regimes.

The Grand River watershed is the largest catchment in southern Ontario. The river's northern and southern sections are influenced by agriculture, whereas central regions receive wastewater effluent and urban runoff. To characterize in-river microbial communities, as they relate to spatial and environmental factors, we conducted two same-day sampling events along the entire 300 km length of the river, representing contrasting flow seasons (high flow spring melt and low flow end of summer).

AnnoView enables large-scale analysis, comparison, and visualization of microbial gene neighborhoods.

The analysis and comparison of gene neighborhoods is a powerful approach for exploring microbial genome structure, function, and evolution. Although numerous tools exist for genome visualization and comparison, genome exploration across large genomic databases or user-generated datasets remains a challenge. Here, we introduce AnnoView, a web server designed for interactive exploration of gene neighborhoods across the bacterial and archaeal tree of life.

Evaluation of multiple displacement amplification for metagenomic analysis of low biomass samples.

Combining multiple displacement amplification (MDA) with metagenomics enables the analysis of samples with extremely low DNA concentrations, making them suitable for high-throughput sequencing. Although amplification bias and nonspecific amplification have been reported from MDA-amplified samples, the impact of MDA on metagenomic datasets is not well understood. We compared three MDA methods (i.

Enigmatic persistence of aerobic methanotrophs in oxygen-limiting freshwater habitats.

Methanotrophic bacteria mitigate emissions of the potent greenhouse gas methane (CH4) from a variety of anthropogenic and natural sources, including freshwater lakes, which are large sources of CH4 on a global scale. Despite a dependence on dioxygen (O2) for CH4 oxidation, abundant populations of putatively aerobic methanotrophs have been detected within microoxic and anoxic waters and sediments of lakes. Experimental work has demonstrated active aerobic methanotrophs under those conditions, but how they are able to persist and oxidize CH4 under O2 deficiency remains enigmatic.

Impact of dry density and incomplete saturation on microbial growth in bentonite clay for nuclear waste storage.

Aims: Many countries are in the process of designing a deep geological repository (DGR) for long-term storage of used nuclear fuel. For several designs, used fuel containers will be placed belowground, with emplacement tunnels being backfilled using a combination of highly compacted powdered bentonite clay buffer boxes surrounded by a granulated "gapfill" bentonite. To limit the potential for microbiologically influenced corrosion of used fuel containers, identifying conditions that suppress microbial growth is critical for sustainable DGR design.

Stable microbial community in compacted bentonite after 5 years of exposure to natural granitic groundwater.

The Materials Corrosion Test (MaCoTe) at the Underground Research Laboratory in Grimsel, Switzerland, assesses the microbiology and corrosion behavior of engineered barrier components of a deep geological repository (DGR) for long-term disposal of high-level nuclear waste. Diversity and temporal changes of bentonite-associated microbial community profiles were assessed under DGR-like conditions for compacted Wyoming MX-80 bentonite (1.25 g/cm and 1.

Evaluating cpn60 for high-resolution profiling of the mammalian skin microbiome and detection of phylosymbiosis.

Despite being the most widely used phylogenetic marker for amplicon-based profiling of microbial communities, limited phylogenetic resolution of the 16S rRNA gene limits its use for studies of host-microbe co-evolution. In contrast, the cpn60 gene is a universal phylogenetic marker with greater sequence variation capable of species-level resolution. This research compared mammalian skin microbial profiles generated from cpn60 and 16S rRNA gene sequencing approaches, testing for patterns of phylosymbiosis that suggest co-evolutionary host-microbe associations.

Simultaneous nitrification, denitrification, and phosphorus removal from municipal wastewater at low temperature.

A lab-scale sequencing batch reactor was employed to study simultaneous nitrification, denitrification, and phosphorus removal (SNDPR) when treating municipal wastewater at 10 °C for 158 days. An anaerobic/aerobic configuration that had previously been effective when treating synthetic wastewater was explored, however, these conditions were relatively ineffective for real municipal wastewater. Incorporation of a post-anoxic phase (i.

DNA-, RNA-, and Protein-Based Stable-Isotope Probing for High-Throughput Biomarker Analysis of Active Microorganisms.

Stable-isotope probing (SIP) enables researchers to target active populations within complex microbial communities, which is achieved by providing growth substrates enriched in heavy isotopes, usually in the form of C, O, or N. After growth on the substrate and subsequent extraction of microbial biomarkers, typically nucleic acids or proteins, the SIP technique is used for the recovery and analysis of isotope-labelled biomarkers from active microbial populations. In the years following the initial development of DNA- and RNA-based SIP, it was common practice to characterize labelled populations by targeted gene analysis.

Large Fractionation in Iron Isotopes Implicates Metabolic Pathways for Iron Cycling in Boreal Shield Lakes.

Stable Fe isotopes have only recently been measured in freshwater systems, mainly in meromictic lakes. Here we report the δFe of dissolved, particulate, and sediment Fe in two small dimictic boreal shield headwater lakes: manipulated eutrophic Lake 227, with annual cyanobacterial blooms, and unmanipulated oligotrophic Lake 442. Within the lakes, the range in δFe is large (ca.

Nitrogen removal pathways during simultaneous nitrification, denitrification, and phosphorus removal under low temperature and dissolved oxygen conditions.

Nitrogen removal pathways of simultaneous nitrification, denitrification, and phosphorus removal (SNDPR) at low dissolved oxygen (0.3 mg/L) and temperature (10℃) were explored to understand nitrogen removal mechanisms. Biological nitrogen and phosphorus removal was sustained with total inorganic nitrogen removal, phosphorus removal, and simultaneous nitrification and denitrification (SND) efficiencies of 62.

Ammonia-oxidizing archaea and complete ammonia-oxidizing in water treatment systems.

Nitrification, the oxidation of ammonia to nitrate via nitrite, is important for many engineered water treatment systems. The sequential steps of this respiratory process are carried out by distinct microbial guilds, including ammonia-oxidizing bacteria (AOB) and archaea (AOA), nitrite-oxidizing bacteria (NOB), and newly discovered members of the genus that conduct complete ammonia oxidation (comammox). Even though all of these nitrifiers have been identified within water treatment systems, their relative contributions to nitrogen cycling are poorly understood.

Fifteen shades of clay: distinct microbial community profiles obtained from bentonite samples by cultivation and direct nucleic acid extraction.

Characterizing the microbiology of swelling bentonite clays can help predict the long-term behaviour of deep geological repositories (DGRs), which are proposed as a solution for the management of used nuclear fuel worldwide. Such swelling clays represent an important component of several proposed engineered barrier system designs and, although cultivation-based assessments of bentonite clay are routinely conducted, direct nucleic acid detection from these materials has been difficult due to technical challenges. In this study, we generated direct comparisons of microbial abundance and diversity captured by cultivation and direct nucleic acid analyses using 15 reference bentonite clay samples.