Biology subjects

Hubert, C. R.

Publications and source records attributed to Hubert, C. R..

9 recordsLinked to original sources

bioRxiv2026

Targeted Depletion of rRNAs and Tobamoviruses Enhances Viral Discovery in Wastewater Metatranscriptomes

Wastewater contains a complex mixture of nucleic acids derived from human cells, microorganisms, diets, and agricultural or industrial inputs. Metatranscriptomics has the potential to broaden the understanding of wastewater viromes, but its utility is constrained by high-abundance RNA molecules that prevent the detection of lower-abundance signals, including human pathogens. Two major sources of this interfering background signal are ribosomal RNAs (rRNAs) from various organisms, and plant-infecting tobamoviruses from human diets, which are often the most abundant RNA viruses in wastewater. These rRNA molecules and RNA genomes can account for upwards of 95% of total RNA sequences, thereby obscuring the detection of lower-abundance viruses of public health importance. This study evaluated whether combined depletion of ribosomal RNAs and genomic RNA from abundant plant tobamoviruses can improve the recovery of underrepresented viral sequences from wastewater. RNA extracts from a large municipal wastewater treatment plant in Calgary, Canada, were processed with or without probe-based depletion of rRNAs and eight abundant tobamoviruses. Metatranscriptomes revealed that depletion reduced rRNA reads by 90%. Reads assigned to tobamoviruses, which accounted for up to 25% of viral reads in untreated samples, were reduced to <2% of viral reads after depletion. This decrease in undesired RNA signal led to an order of magnitude increase in viral read signals and improved assembled viral genome quality. Furthermore, the number of reads assigned to human and animal pathogens increased 42-fold after depletion, enabling the detection of important pathogens like influenza, RSV, and hepatitis A, which were undecteable in untreated samples. Together, these findings demonstrate that targeted depletion of abundant background RNAs improves the detection of low abundance viruses, and enhances the utility of metatranscriptomic approaches for wastewater viral surveillance.

microbiology↗

bioRxiv2024

Hydrocarbon-degrading microbial populations in permanently cold deep-sea sediments in the NW Atlantic

Permanently cold deep-sea sediments (2500-3500 m water depth) with or without indications of thermogenic hydrocarbon seepage were exposed to naphtha to examine the presence and potential of aerobic hydrocarbon-degrading microbial populations. Monitoring these microcosms for volatile hydrocarbons by GC-MS revealed sediments without in situ hydrocarbons responded more rapidly to naphtha amendment than hydrocarbon seep sediments overall, but seep sediments removed BTEX compounds more readily. Naphtha-driven aerobic respiration was more evident in surface sediment (0-20 cmbsf) than deeper anoxic layers (>130 cmbsf) that responded less rapidly. In all cases, enrichment of Gammaproteobacteria included lineages of Oleispira, Pseudomonas, and Alteromonas known to be associated with marine oil spills. On the other hand, taxa known to be prevalent in situ and diagnostic for thermogenic hydrocarbon seepage in deep sea sediment did not respond to naphtha amendment. This suggests a limited role for seep-associated populations in the context of oil spill biodegradation.

microbiology↗

bioRxiv2024

Denitrification genotypes of endospore-forming Bacillota

Denitrification is a key metabolic process in the global nitrogen cycle and is performed by taxonomically diverse microorganisms. Despite the widespread importance of this metabolism, challenges remain in identifying denitrifying populations and predicting their metabolic end-products based on their genotype. Here, genome-resolved metagenomics was used to explore the denitrification genotype of Bacillota enriched in nitrate-amended high temperature incubations with confirmed N2O and N2 production. A set of 12 hidden Markov models (HMMs) was created to target the diversity of denitrification genes in members of the phylum Bacillota. Genomic potential for complete denitrification was found in five metagenome-assembled genomes (MAGs) from nitrate-amended enrichments, including two novel members of the Brevibacillaceae family. Genomes of complete denitrifiers encode N2O reductase (nos) gene clusters with clade II-type nosZ and often include multiple variants of the nitric oxide reductase (nor) gene. The HMM set applied to all genomes of Bacillota from the Genome Taxonomy Database (GTDB) identified 17 genera inferred to contain complete denitrifiers based on their gene content. Among complete denitrifiers it was common for three distinct nitric oxide reductases to be present (qNOR, bNOR and sNOR) which may reflect the metabolic adaptability of Bacillota in environments with variable redox conditions.

microbiology↗

bioRxiv2024

Novel oil-associated bacteria in Arctic seawater exposed to different nutrient biostimulation regimes

The Arctic Ocean is an oligotrophic ecosystem facing escalating threats of oil spills as ship traffic increases owing to climate change-induced sea ice retreat. Biostimulation is an oil spill mitigation strategy that involves introducing bioavailable nutrients to enhance crude oil biodegradation by endemic oil-degrading microbes. For bioremediation to offer a viable response for future oil spill mitigation in extreme Arctic conditions, a better understanding of the effects of nutrient addition on Arctic marine microorganisms is needed. Comprehensive population tracking of controlled oil-spill microcosms using cell counting and microbial biodiversity screening revealed a significant decline in community diversity together with changes in microbial community composition. These shifts were also indicative of changes in prevailing genomic traits as inferred from 16S rRNA taxonomy of resulting communities. In addition to well-recognized hydrocarbonoclastic bacteria, differential abundance analysis highlighted significant enrichment of unexpected genera Lacinutrix, Halarcobacter and Candidatus Pseudothioglobus. These groups have not been associated with hydrocarbon biodegradation until now, even though genomes from closely related isolates confirm the potential for hydrocarbon metabolism. These findings broaden understanding of marine oil spill bioremediation and how Arctic marine microbiomes and their novel lineages can respond to nutrient biostimulation as a strategy for oil spill mitigation. ImportanceA comprehensive characterization and understanding of the impact of marine bioremediation strategies in the Arctic is crucial for effectively managing oil contamination. Such understanding enables an evaluation of the ecological impacts associated with mitigation strategies to minimize negative effects on sensitive ecosystems. By introducing external nutrients into areas affected by spills, microbial growth can be stimulated, enhancing hydrocarbon degradation by naturally occurring oil-degrading microorganisms. This may include novel microbial groups in permanently cold Arctic waters, where fewer oil biodegradation studies have been performed. It is also important to consider how nutrient addition may affect endemic microbial communities after successful remediation has occurred in oil-contaminated zones. Promoting naturally occurring oil-degrading microorganisms may have significant implications on nutrient cycling and marine food webs, which are critical for sustaining the health and well-being of coastal Indigenous communities in northern latitudes.

microbiology↗

bioRxiv2023

Polymer biodegradation by Halanaerobium promotes reservoir souring during hydraulic fracturing.

Hydraulically fractured shale reservoirs have facilitated studies of unexplored niches in the continental deep biosphere. Members of the genus Halanaerobium are ubiquitous in these environments. Polymers like guar gum used as gelling agents in hydraulic fracturing fluids are known to be fermentable substrates, but metabolic pathways encoding these processes have not been characterized. To explore this, produced water samples from the Permian Basin were incubated at 30{degrees}C to simulate above-ground storage conditions, and at 60{degrees}C to simulate subsurface reservoir conditions. Guar metabolism coincided with Halanaerobium enrichment only at 30{degrees}C, revealing genes for polymer biodegradation through the mixed-acid fermentation pathway in different metagenome-assembled genomes (MAGs). Whereas thiosulfate reduction to sulfide is often invoked to explain the dominance of Halanaerobium in these settings, Halanaerobium genomes did not uncover genes for this metabolism. Sulfide production was observed in 60{degrees}C incubations, with corresponding enrichment of Desulfohalobium and Desulfovibrionaceae that possess complete pathways for coupling mannose and acetate oxidation to sulfate reduction. These findings outline how production of fermentation intermediates (mannose, acetate) by Halanaerobium in topside settings can result in reservoir souring when these metabolites are introduced into the subsurface through produced water re-use. ImportanceHydraulically fractured shale oil reservoirs are ideal for studying extremophiles such as thermohalophiles. During hydraulic fracturing, reservoir production water is stored in surface ponds prior to re-use. Microorganisms in these systems therefore need to withstand various environmental changes such as the swing between warm downhole oil reservoir temperatures and cooler surface conditions. This study follows this water cycle during fracking and the associated microbial metabolic potential. Of particular interest are members of the genus Halanaerobium, that have been reported to reduce thiosulfate contributing to souring of oil reservoirs. Here, we show that some Halanaerobium strains were unable to grow under oil reservoir temperatures and do not possess genes for thiosulfate reduction. Rather, it is likely that these organisms metabolize complex organics in fracking fluids at lower temperatures, thereby generating substrates that support reservoir souring by thermophilic sulfate-reducing bacteria at higher temperatures.

microbiology↗

bioRxiv2022

SituSeq: An offline protocol for rapid and remote Nanopore amplicon sequence analysis

Microbiome analysis through 16S rRNA gene sequencing is a crucial tool for understanding the microbial ecology of any habitat or ecosystem. However, workflows require large equipment, stable internet, and extensive computing power such that most of the work is performed far away from sample collection in both space and time. Performing amplicon sequencing and analysis at sample collection would have positive implications in many instances including remote fieldwork and point-of-care medical diagnoses. Here we present SituSeq, an offline and portable workflow for the sequencing and analysis of 16S rRNA gene amplicons using the Nanopore MinION and a standard laptop computer. SituSeq was validated using the same environmental DNA to sequence Nanopore 16S rRNA gene amplicons, Illumina 16S rRNA gene amplicons, and Illumina metagenomes. Comparisons revealed consistent community composition, ecological trends, and sequence identity across platforms. Correlation between the abundance of phyla in Illumina and Nanopore data sets was high (Pearsons r = 0.9), and over 70% of Illumina 16S rRNA gene sequences matched a Nanopore sequence with greater than 97% sequence identity. On board a research vessel on the open ocean, SituSeq was used to analyze amplicon sequences from deep sea sediments less than two hours after sequencing, and eight hours after sample collection. The rapidly available results informed decisions about subsequent sampling in near real-time while the offshore expedition was still underway. SituSeq is a portable and robust workflow that helps to bring the power of microbial genomics and diagnostics to many more researchers and situations.

microbiology↗

bioRxiv2022

Bacterial anomalies in seabed sediments associated with deep water hydrocarbon seepage

Deep sea hydrocarbon seep detection relies predominantly on geochemical analyses of seabed marine sediment cores to identify the presence of gas or oil. The presence of seeping hydrocarbons in these locations alters resident microbial community structure, leading to culture-based biodegradation assays as a complement to geochemical tools for seep detection. Biodiversity surveys of microbial communities can offer a similar proxy for seeping hydrocarbons, but this strategy has not been extensively investigated in deep water settings. In this study, 16S rRNA gene sequencing of bacterial communities was performed on sediment cores obtained in >2500 m water depth at 43 different locations in the NW Atlantic Ocean. Core samples from as deep as 10 metres below seafloor (mbsf) were assessed for gas composition, gas isotopes and liquid hydrocarbons. Over 650 bacterial 16S rRNA gene amplicon libraries were constructed from different sediment depths at these locations. Select sites showed strong evidence for the presence of thermogenic or biogenic hydrocarbons such that bacterial population analyses revealed significant differences between hydrocarbon seep and non-seep locations. Specific bacterial indicators were associated with different sediment depth intervals. Caldatribacteriota and Campilobacterota OTUs were observed in high relative sequence abundance in hydrocarbon seep sediments, particularly in the 20-50 cmbsf interval. Furthermore, these groups were differentially abundant between sites with thermogenic and biogenic hydrocarbons. The patterns revealed here suggest that microbial screening has the potential to play a key role in hydrocarbon seep detection and characterisation in remote deep-sea environments.

microbiology↗

bioRxiv2021

Geological processes mediate a subsurface microbial loop in the deep biosphere

Summary paragraphThe deep biosphere is the largest microbial habitat on Earth and features abundant bacterial endospores1,2. Whereas dormancy and survival at theoretical energy minima are hallmarks of subsurface microbial populations3, the roles of fundamental ecological processes like dispersal and selection in these environments are poorly understood4. Here we combine geophysics, geochemistry, microbiology and genomics to investigate biogeography in the subsurface, focusing on bacterial endospores in a deep-sea setting characterized by thermogenic hydrocarbon seepage. Thermophilic endospores in permanently cold seabed sediments above petroleum seep conduits were correlated with the presence of hydrocarbons, revealing geofluid-facilitated cell migration pathways originating in deep oil reservoirs. Genomes of thermophilic bacteria highlight adaptations to life in anoxic petroleum systems and reveal that these dormant populations are closely related to oil reservoir microbiomes from around the world. After transport out of the subsurface and into the deep-sea, thermophilic endospores re-enter the geosphere by sedimentation. Viable thermophilic endospores spanning the top several metres of the seabed correspond with total endospore counts that are similar to or exceed the global average. Burial of dormant cells enables their environmental selection in sedimentary formations where new petroleum systems establish, completing a geological microbial loop that circulates living biomass in and out of the deep biosphere.

microbiology↗

bioRxiv2021

Extremely thermophilic endospores germinate and metabolise organic carbon in sediments heated to above 80°C

Endospores of thermophilic bacteria are widespread in cold seabed environments where they remain dormant during initial burial in accumulating sediments. The temperature increase during sedimentation can be simulated in experimental heating of sediments, resulting in the temperature-dependent activation of different endospore populations from the microbial seed bank. Here we investigated the response of endospore populations to heating at extreme high temperature (80- 99{degrees}C). Metabolites for germination and organic matter degradation (dipicolinic acid and organic acids) revealed both endospore germination and subsequent metabolism at [≥]80{degrees}C. Endospore-forming Firmicutes with the genomic potential for organic carbon and nitrogen transformation were recovered by genome-resolved metagenomics. Genomes from Symbiobacteriales, Thermosediminibacteriales, Moorellales and Calditerricolales encode multiple mechanisms for high temperature degradation of sedimentary organic carbon and features of necromass that accumulate during sediment burial including saccharides, amino and nucleic acids. The results provide insight into the metabolism of novel carbon cycling microorganisms activated at high temperature, and suggest that extremely thermophilic Firmicutes dispersed in the ocean are poised to germinate in response to sediment heating during burial and transform a wide range of organic substrates.

microbiology↗