Here we assess the ability of random whole metagenomic sequencing approaches to discriminate between similar soils from two geographically distinct urban sites for application in forensic science. ...Repeat samples from two parklands in residential areas separated by approximately 3 km were collected and the DNA was extracted. Shotgun, whole genome amplification (WGA) and single arbitrarily primed DNA amplification (AP-PCR) based sequencing techniques were then used to generate soil metagenomic profiles. Full and subsampled metagenomic datasets were then annotated against M5NR/M5RNA (taxonomic classification) and SEED Subsystems (metabolic classification) databases. Further comparative analyses were performed using a number of statistical tools including: hierarchical agglomerative clustering (CLUSTER); similarity profile analysis (SIMPROF); non-metric multidimensional scaling (NMDS); and canonical analysis of principal coordinates (CAP) at all major levels of taxonomic and metabolic classification. Our data showed that shotgun and WGA-based approaches generated highly similar metagenomic profiles for the soil samples such that the soil samples could not be distinguished accurately. An AP-PCR based approach was shown to be successful at obtaining reproducible site-specific metagenomic DNA profiles, which in turn were employed for successful discrimination of visually similar soil samples collected from two different locations.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Plastic production continually increases its share of global oil consumption. Thermoplastic elastomers (TPEs) are a necessary component of many industries, from automotive and construction to ...healthcare and medical devices. To reduce the environmental burden of TPE production on the world, we developed two new ABA triblock copolymers synthesized through cationic reversable addition–fragmentation chain transfer (RAFT) polymerization from renewable monomers. Using poly(isobutyl vinyl ether) (PIBVE) as the soft block and either poly( p -methoxystyrene) (PMOS) or poly(2,3-dihydrofuran) (PDHF) as the hard blocks, we produced triblock copolymers with varying volume fractions and characterized their material properties. PDHF-PIBVE-PDHF is sourced almost entirely from simple alcohols and exhibits mechanical properties comparable to those of commercial TPEs. This effort demonstrates the utility of cationic RAFT for the production of sustainable TPEs.
Many archaeological science studies use the concept of "provenance", where the origins of cultural material can be determined through physical or chemical properties that relate back to the origins ...of the material. Recent studies using DNA profiling of bacteria have been used for the forensic determination of soils, towards determination of geographic origin. This manuscript presents a novel approach to the provenance of archaeological minerals and related materials through the use of 16S rRNA sequencing analysis of microbial DNA. Through the microbial DNA characterization from ochre and multivariate statistics, we have demonstrated the clear discrimination between four distinct Australian cultural ochre sites.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The rapid expansion of 16S rRNA gene sequencing in challenging clinical contexts has resulted in a growing body of literature of variable quality. To a large extent, this is due to a failure to ...address spurious signal that is characteristic of samples with low levels of bacteria and high levels of non-bacterial DNA. We have developed a workflow based on the paired-end read Illumina MiSeq-based approach, which enables significant improvement in data quality, post-sequencing. We demonstrate the efficacy of this methodology through its application to paediatric upper-respiratory samples from several anatomical sites.
A workflow for processing sequence data was developed based on commonly available tools. Data generated from different sample types showed a marked variation in levels of non-bacterial signal and 'contaminant' bacterial reads. Significant differences in the ability of reference databases to accurately assign identity to operational taxonomic units (OTU) were observed. Three OTU-picking strategies were trialled as follows: de novo, open-reference and closed-reference, with open-reference performing substantially better. Relative abundance of OTUs identified as potential reagent contamination showed a strong inverse correlation with amplicon concentration allowing their objective removal. The removal of the spurious signal showed the greatest improvement in sample types typically containing low levels of bacteria and high levels of human DNA. A substantial impact of pre-filtering data and spurious signal removal was demonstrated by principal coordinate and co-occurrence analysis. For example, analysis of taxon co-occurrence in adenoid swab and middle ear fluid samples indicated that failure to remove the spurious signal resulted in the inclusion of six out of eleven bacterial genera that accounted for 80% of similarity between the sample types.
The application of the presented workflow to a set of challenging clinical samples demonstrates its utility in removing the spurious signal from the dataset, allowing clinical insight to be derived from what would otherwise be highly misleading output. While other approaches could potentially achieve similar improvements, the methodology employed here represents an accessible means to exclude the signal from contamination and other artefacts.
Drought events and the overexploitation of freshwater resources have led to the increased need to manage groundwater reserves. Aquifer storage and recovery (ASR), whereby artificial water is injected ...into aquifers for storage, is one of the proposed methods by which freshwater supplies can be increased. Microbial clogging following injection, however, is a major issue. Here, during laboratory simulations of ASR, we used flow cytometry and bar-coded pyrosequencing to investigate changes in microbial abundance and community dynamics. Bacterial abundance ranged from 5.0 × 104 to 1.4 × 107 cells ml-1 before the addition of synthetic wastewater. Following wastewater addition, a 25-fold decrease in abundance was observed, coinciding with a 12-fold increase in viral abundance. Taxa shifted from an overrepresentation of Sphingomonadales, Sphingobacteriales, Rhodospirillales, Caulobacterales, Legionellales, Bacillales, Fusobacteriales and Verrucomicrobiales prior to the addition of synthetic wastewater to Burkholderiales, Actinomycetales, Pseudomonadales, Xanthomonadales, Rhodobacterales, Thizobiales and Thiotrichales following the addition of synthetic wastewater. Furthermore, a significant difference in overall taxonomic composition between the groundwater samples before and after the addition of synthetic wastewater was observed, with water samples exhibiting more similarity to sediment samples after wastewater was added. Collectively, these results suggest that ASR may alter the taxonomic composition of endemic microbial communities and that complete profiles of groundwater properties, including microbial community abundance and composition need to be taken into consideration when selecting aquifers for ASR practices.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The functional dynamics of microbial communities are largely responsible for the clean-up of hydrocarbons in the environment. However, knowledge of the distinguishing functional genes, known as the ...metabolic footprint, present in hydrocarbon-impacted sites is still scarcely understood. Here, we conducted several multivariate analyses to characterise the metabolic footprints present in a variety of hydrocarbon-impacted and non-impacted sediments. Non-metric multi-dimensional scaling (NMDS) and canonical analysis of principal coordinates (CAP) showed a clear distinction between the two groups. A high relative abundance of genes associated with cofactors, virulence, phages and fatty acids were present in the non-impacted sediments, accounting for 45.7% of the overall dissimilarity. In the hydrocarbon-impacted sites, a high relative abundance of genes associated with iron acquisition and metabolism, dormancy and sporulation, motility, metabolism of aromatic compounds and cell signalling were observed, accounting for 22.3% of the overall dissimilarity. These results suggest a major shift in functionality has occurred with pathways essential to the degradation of hydrocarbons becoming overrepresented at the expense of other, less essential metabolisms.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The spatial distributions of organism abundance and diversity are often heterogeneous. This includes the sub-centimetre distributions of microbes, which have 'hotspots' of high abundance, and ...'coldspots' of low abundance. Previously we showed that 300 μl abundance hotspots, coldspots and background regions were distinct at all taxonomic levels. Here we build on these results by showing taxonomic micropatches within these 300 μl microscale hotspots, coldspots and background regions at the 1 μl scale. This heterogeneity among 1 μl subsamples was driven by heightened abundance of specific genera. The micropatches were most pronounced within hotspots. Micropatches were dominated by Pseudomonas, Bacteroides, Parasporobacterium and Lachnospiraceae incertae sedis, with Pseudomonas and Bacteroides being responsible for a shift in the most dominant genera in individual hotspot subsamples, representing up to 80.6% and 47.3% average abundance, respectively. The presence of these micropatches implies the ability these groups have to create, establish themselves in, or exploit heterogeneous microenvironments. These genera are often particle-associated, from which we infer that these micropatches are evidence for sub-millimetre aggregates and the aquatic polymer matrix. These findings support the emerging paradigm that the microscale distributions of planktonic microbes are numerically and taxonomically heterogeneous at scales of millimetres and less. We show that microscale microbial hotspots have internal structure within which specific local nutrient exchanges and cellular interactions might occur.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Rising atmospheric CO₂, cₐ, is expected to affect stomatal regulation of leaf gas‐exchange of woody plants, thus influencing energy fluxes as well as carbon (C), water, and nutrient cycling of ...forests. Researchers have proposed various strategies for stomatal regulation of leaf gas‐exchange that include maintaining a constant leaf internal CO₂, cᵢ, a constant drawdown in CO₂ (cₐ − cᵢ), and a constant cᵢ/cₐ. These strategies can result in drastically different consequences for leaf gas‐exchange. The accuracy of Earth systems models depends in part on assumptions about generalizable patterns in leaf gas‐exchange responses to varying cₐ. The concept of optimal stomatal behavior, exemplified by woody plants shifting along a continuum of these strategies, provides a unifying framework for understanding leaf gas‐exchange responses to cₐ. To assess leaf gas‐exchange regulation strategies, we analyzed patterns in cᵢ inferred from studies reporting C stable isotope ratios (δ¹³C) or photosynthetic discrimination (∆) in woody angiosperms and gymnosperms that grew across a range of cₐ spanning at least 100 ppm. Our results suggest that much of the cₐ‐induced changes in cᵢ/cₐ occurred across cₐ spanning 200 to 400 ppm. These patterns imply that cₐ − cᵢ will eventually approach a constant level at high cₐ because assimilation rates will reach a maximum and stomatal conductance of each species should be constrained to some minimum level. These analyses are not consistent with canalization toward any single strategy, particularly maintaining a constant cᵢ. Rather, the results are consistent with the existence of a broadly conserved pattern of stomatal optimization in woody angiosperms and gymnosperms. This results in trees being profligate water users at low cₐ, when additional water loss is small for each unit of C gain, and increasingly water‐conservative at high cₐ, when photosystems are saturated and water loss is large for each unit C gain.
A metagenomic analysis of two aquifer systems located under a dairy farming region was performed to examine to what extent the composition and function of microbial communities varies between ...confined and surface‐influenced unconfined groundwater ecosystems. A fundamental shift in taxa was seen with an overrepresentation of Rhodospirillales, Rhodocyclales, Chlorobia and Circovirus in the unconfined aquifer, while Deltaproteobacteria and Clostridiales were overrepresented in the confined aquifer. A relative overrepresentation of metabolic processes including antibiotic resistance (β‐lactamase genes), lactose and glucose utilization and DNA replication were observed in the unconfined aquifer, while flagella production, phosphate metabolism and starch uptake pathways were all overrepresented in the confined aquifer. These differences were likely driven by differences in the nutrient status and extent of exposure to contaminants of the two groundwater systems. However, when compared with freshwater, ocean, sediment and animal gut metagenomes, the unconfined and confined aquifers were taxonomically and metabolically more similar to each other than to any other environment. This suggests that intrinsic features of groundwater ecosystems, including low oxygen levels and a lack of sunlight, have provided specific niches for evolution to create unique microbial communities. Obtaining a broader understanding of the structure and function of microbial communities inhabiting different groundwater systems is particularly important given the increased need for managing groundwater reserves of potable water.
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