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.e. bulk MDA, emulsion MDA, and primase MDA) for metagenomic analysis of two DNA template concentrations (approx. 1 and 100 pg) derived from a microbial community standard "mock community" and two low biomass environmental samples (i.e. borehole fluid and groundwater). We assessed the impact of MDA on metagenome-based community composition, assembly quality, functional profiles, and binning. We found amplification bias against high GC content genomes but relatively low nonspecific amplification such as chimeras, artifacts, or contamination for all MDA methods. We observed MDA-associated representational bias for microbial community profiles, especially for low-input DNA and with the primase MDA method. Nevertheless, similar taxa were represented in MDA-amplified libraries to those of unamplified samples. The MDA libraries were highly fragmented, but similar functional profiles to the unamplified libraries were obtained for bulk MDA and emulsion MDA at higher DNA input and across these MDA libraries for the groundwater sample. Medium to low-quality bins were possible for the high input bulk MDA metagenomes for the most simple microbial communities, borehole fluid, and mock community. Although MDA-based amplification should be avoided, it can still reveal meaningful taxonomic and functional information from samples with extremely low DNA concentration where direct metagenomics is otherwise impossible.

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. This study investigated microbial communities in powdered and gapfill bentonite clay incubated in oxic pressure vessels at dry densities between 1.1 g cm-3 (i.e. below repository target) and 1.6 g cm-3 (i.e. at or above repository target) as a 1-year time series. RESULTS: Our results showed an initial (i.e. 1 month) increase in the abundance of culturable heterotrophs associated with all dry densities <1.6 g cm-3, which reveals growth during transient low-pressure conditions associated with the bentonite saturation process. Following saturation, culturable heterotroph abundances decreased to those of starting material by the 6-month time point for all 1.4 and 1.6 g cm-3 pressure vessels, and the most probable numbers of culturable sulfate-reducing bacteria (SRB) remained constant for all vessels and time points. The 16S rRNA gene sequencing results showed a change in microbial community composition from the starting material to the 1-month time point, after which time most samples were dominated by sequences associated with Pseudomonas, Bacillus, Cupriavidus, and Streptomyces. Similar taxa were identified as dominant members of the culture-based community composition, demonstrating that the dominant members of the clay microbial communities are viable. Members of the spore-forming Desulfosporosinus genus were the dominant SRB for both clay and culture profiles. CONCLUSIONS: After initial microbial growth while bentonite was below target pressure in the early phases of saturation, microbial growth in pressure vessels with dry densities of at least 1.4 g cm-3 was eventually suppressed as bentonite neared saturation.

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/cm3 and 1.50 g/cm3 targeted dry densities) exposed to natural groundwater. Using culture-dependent and molecular techniques, samples taken from the outside layer of 5-year borehole modules revealed up to 66% and 23% of 16S rRNA gene sequences affiliated with Desulfosporosinus and Desulfovibrio, respectively. Putatively involved in sulfate reduction, these taxa were almost undetectable within the bentonite core. Instead, microbial profiles of the inner bentonite core were similar to uncompacted bentonite used to pack modules years earlier, and were consistent with a previously published 1-year time point, revealing no detectable microbial growth. Abundances of culturable aerobic and anaerobic heterotrophic bacteria in the uncompacted bentonite were relatively low, with less than 1,000 and 100 colony-forming units (CFUs) per gram dry weight, respectively. Nearly 5 years after emplacement, culturable heterotrophic bacterial CFUs and sulfate-reducing bacteria did not change significantly inside the bentonite core. Phospholipid fatty acid data indicated similar lipid abundance, and corresponding cell abundance estimates, for inner 5-year MaCoTe bentonite samples compared to those previously obtained for 1-year incubations. Collectively, our results provide complementary evidence for microbial stability inside highly compacted bentonite exposed to conditions that mimic engineered barrier components of a deep geological repository. IMPORTANCE The long-term safety of a deep geological repository for used nuclear fuel is dependent on the performance of the engineered and natural barriers. Microbial activity can produce chemical species that can influence the corrosion of the disposal containers for used nuclear fuel. Although previous studies have evaluated the microbiology of compacted bentonite clay within subsurface environments, these have been limited to relatively short incubations (i.e., 1 year). The current study provides a unique 5-year perspective that reinforces previous findings of growth inhibition for bentonite clay exposed to in situ subsurface conditions.

A Coarse-Grained Interaction Model for Sodium Dominant Montmorillonite.

Montmorillonite is the main crystalline mineral present in bentonite. It is an absorbent, swelling material; the physical chemistry underlying its ability to absorb water and swell occurs at the nanoscale, governed by electrical double-layer interactions. In turn, absorption and swelling lead to important changes in the macroscopic transport properties of the clay. Mesoscale models can help us establish a link between these nanoscale processes and macroscale properties, notably by providing a detailed description of its pore network. Models on the scale of hundreds to thousands of nanometers are required, which cannot realistically be handled using traditional all-atom molecular dynamics simulations. This work presents a coarse-grained (CG) mesoscale model of sodium montmorillonite. In our model, montmorillonite platelets are represented by two types of particles: central nonhydrogen-bonded particles and edge hydrogen-bonding particles. The particle interactions are described by two-body potentials, which were optimized based on all-atom molecular dynamics simulations. Specifically, several potential mean force calculations involving dry and hydrated montmorillonite were performed, using the ClayFF potential to calculate interatomic forces. The CG model was validated by testing the scalability of the model, testing its ability to reproduce potentials of mean force reported elsewhere in the literature, and by comparing the calculated elastic properties of a system containing 1000 Na montmorillonite platelets to experimentally measured elastic properties of bentonite. The simulated elastic properties obtained using our mesoscale model agree with these experimental values.

Microbiology of barrier component analogues of a deep geological repository.

Canada is currently implementing a site selection process to identify a location for a deep geological repository (DGR) for the long-term storage of Canada's used nuclear fuel, wherein used nuclear fuel bundles will be sealed inside copper-coated carbon steel containers, encased in highly compacted bentonite clay buffer boxes, and sealed deep underground in a stable geosphere. Because a DGR must remain functional for a million years, it is important to examine ancient natural systems that serve as analogues for planned DGR components. Specifically, studying the microbiology of natural analogue components of a DGR is important for developing an understanding of the types of microorganisms that may be able to grow and influence the long-term stability of a DGR. This study explored the abundance, viability, and composition of microorganisms in several ancient natural analogues using a combination of cultivation and cultivation-independent approaches. Samples were obtained from the Tsukinuno bentonite deposit (Japan) that formed ∼10 mya, the Opalinus Clay formation (Switzerland) that formed ∼174 mya, and Canadian shield crystalline rock from Northern Ontario that formed ∼2.7 bya. Analysis of 16S rRNA gene amplicons revealed that three of the ten Tsukinuno bentonite samples analyzed were dominated by putative aerobic heterotrophs and fermenting bacteria from the phylum Actinobacteria, whereas five of the Tsukinuno bentonite samples were dominated by sequences associated with putative acidophilic chemolithoautotrophs capable of sulfur reduction. The remaining Tsukinuno bentonite samples, the Northern Ontario rock samples, and the Opalinus Clay samples generated inconsistent replicate 16S rRNA gene profiles and were associated primarily with contaminant sequences, suggesting that the microbial profiles detected were not sample-specific but spurious. Culturable aerobic heterotroph abundances were relatively low for all Tsukinuno bentonite samples, culturable anaerobic heterotrophs were only detected in half of the Tsukinuno samples, and sulfate-reducing bacteria (SRB) were only detected in one Tsukinuno sample by cultivation. Culture-specific 16S rRNA gene profiles from Tsukinuno clay samples demonstrated the presence of phyla Bacteroidetes, Proteobacteria, Actinobacteria, and Firmicutes among aerobic heterotroph cultures and additional bacteria from the phyla Actinobacteria and Firmicutes from anaerobic heterotroph plate incubations. Only one nucleic acid sequence detected from a culture was also associated with its corresponding clay sample profile, suggesting that nucleic acids from culturable bacteria were relatively rare within the clay samples. Sequencing of DNA extracted from the SRB culture revealed that the taxon present in the culture was affiliated with the genus Desulfosporosinus, which has been found in related bentonite clay analyses. Although the crystalline rock and Opalinus Clay samples were associated with inconsistent, likely spurious 16S rRNA gene profiles, we show evidence for viable and detectable microorganisms within several Tsukinuno natural analogue bentonite samples.

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. Regardless of clay starting material, the corresponding profiles from cultivation-based approaches were consistently associated with phylogenetically similar sulfate-reducing bacteria, denitrifiers, aerobic heterotrophs, and fermenters, demonstrating that any DGR-associated growth may be consistent, regardless of the specific bentonite clay starting material selected for its construction. Furthermore, dominant nucleic acid sequences in the as-received clay microbial profiles did not correspond with the bacteria that were enriched or isolated in culture. Few core taxa were shared among cultivation and direct nucleic acid analysis profiles, yet those in common were primarily affiliated with Streptomyces, Micrococcaceae, Bacillus, and Desulfosporosinus genera. These putative desiccation-resistant bacteria associated with diverse bentonite clay samples can serve as targets for experiments that evaluate microbial viability and growth within DGR-relevant conditions. Our data will be important for global nuclear waste management organizations, demonstrating that identifying appropriate design conditions with suitable clay swelling properties will prevent growth of the same subset of clay-associated bacteria, regardless of clay origin or processing conditions.

Determination of the local corrosion rate of magnesium alloys using a shear force mounted scanning microcapillary method.

The successful development of scanning probe techniques to characterize corrosion in situ using multifunctional probes is intrinsically tied to surface topography signal decoupling from the measured electrochemical fluxes. One viable strategy is the shear force controlled scanning microcapillary method. Using this method, pulled quartz micropipettes with an aperture of 500 nm diameter were used to resolve small and large variations in topography in order to quantify the local corrosion rate of microgalvanically and galvanically corroded Mg alloys. To achieve topography monitoring of corroded surfaces, shear force feedback was employed to position the micropipette at a reproducible working height above the substrate. We present proof of concept measurements over a galvanic couple of a magnesium alloy (AE44) and mild steel along with a microgalvanically corroded ZEK100 Mg alloy, which illustrates the ability of shear force to track small (1.4 µm) and large (700 µm) topographic variations from high aspect ratio features. Furthermore, we demonstrate the robustness of the technique by acquiring topographic data for 4 mm along the magnesium-steel galvanic couple sample and a 250 × 30 µm topography map over the ZEK100 Mg alloy. All topography results were benchmarked using standard optical microscopies (profilometry and confocal laser scanning microscopy).