Trace gas oxidation sustains energy needs of a thermophilic archaeon at suboptimal temperatures.

Diverse aerobic bacteria use atmospheric hydrogen (H2) and carbon monoxide (CO) as energy sources to support growth and survival. Such trace gas oxidation is recognised as a globally significant process that serves as the main sink in the biogeochemical H2 cycle and sustains microbial biodiversity in oligotrophic ecosystems. However, it is unclear whether archaea can also use atmospheric H2. Here we show that a thermoacidophilic archaeon, Acidianus brierleyi (Thermoproteota), constitutively consumes H2 and CO to sub-atmospheric levels. Oxidation occurs across a wide range of temperatures (10 to 70 °C) and enhances ATP production during starvation-induced persistence under temperate conditions. The genome of A. brierleyi encodes a canonical CO dehydrogenase and four distinct [NiFe]-hydrogenases, which are differentially produced in response to electron donor and acceptor availability. Another archaeon, Metallosphaera sedula, can also oxidize atmospheric H2. Our results suggest that trace gas oxidation is a common trait of Sulfolobales archaea and may play a role in their survival and niche expansion, including during dispersal through temperate environments.

A genus in the bacterial phylum Aquificota appears to be endemic to Aotearoa-New Zealand.

Allopatric speciation has been difficult to examine among microorganisms, with prior reports of endemism restricted to sub-genus level taxa. Previous microbial community analysis via 16S rRNA gene sequencing of 925 geothermal springs from the Taupo Volcanic Zone (TVZ), Aotearoa-New Zealand, revealed widespread distribution and abundance of a single bacterial genus across 686 of these ecosystems (pH 1.2-9.6 and 17.4-99.8 °C). Here, we present evidence to suggest that this genus, Venenivibrio (phylum Aquificota), is endemic to Aotearoa-New Zealand. A specific environmental niche that increases habitat isolation was identified, with maximal read abundance of Venenivibrio occurring at pH 4-6, 50-70 °C, and low oxidation-reduction potentials. This was further highlighted by genomic and culture-based analyses of the only characterised species for the genus, Venenivibrio stagnispumantis CP.B2T, which confirmed a chemolithoautotrophic metabolism dependent on hydrogen oxidation. While similarity between Venenivibrio populations illustrated that dispersal is not limited across the TVZ, extensive amplicon, metagenomic, and phylogenomic analyses of global microbial communities from DNA sequence databases indicates Venenivibrio is geographically restricted to the Aotearoa-New Zealand archipelago. We conclude that geographic isolation, complemented by physicochemical constraints, has resulted in the establishment of an endemic bacterial genus.

Draft genome sequence of Thermococcus waiotapuensis WT1T, a thermophilic sulfur-dependent archaeon from the order Thermococcales.

Thermococcus waiotapuensis WT1T is a thermophilic, peptide, and amino acid-fermenting archaeon from the order Thermococcales. It was isolated from Waiotapu, Aotearoa-New Zealand, and has a genome size of 1.80 Mbp. The genome contains 2,000 total genes, of which 1,913 encode proteins and 46 encode tRNA.

Thermophilic methane oxidation is widespread in Aotearoa-New Zealand geothermal fields.

Geothermal areas represent substantial point sources for greenhouse gas emissions such as methane. While it is known that methanotrophic microorganisms act as a biofilter, decreasing the efflux of methane in most soils to the atmosphere, the diversity and the extent to which methane is consumed by thermophilic microorganisms in geothermal ecosystems has not been widely explored. To determine the extent of biologically mediated methane oxidation at elevated temperatures, we set up 57 microcosms using soils from 14 Aotearoa-New Zealand geothermal fields and show that moderately thermophilic (>40°C) and thermophilic (>60°C) methane oxidation is common across the region. Methane oxidation was detected in 54% (n = 31) of the geothermal soil microcosms tested at temperatures up to 75°C (pH 1.5-8.1), with oxidation rates ranging from 0.5 to 17.4 µmol g-1 d-1 wet weight. The abundance of known aerobic methanotrophs (up to 60.7% Methylacidiphilum and 11.2% Methylothermus) and putative anaerobic methanotrophs (up to 76.7% Bathyarchaeota) provides some explanation for the rapid rates of methane oxidation observed in microcosms. However, not all methane oxidation was attributable to known taxa; in some methane-consuming microcosms we detected methanotroph taxa in conditions outside of their known temperature range for growth, and in other examples, we observed methane oxidation in the absence of known methanotrophs through 16S rRNA gene sequencing. Both of these observations suggest unidentified methane oxidizing microorganisms or undescribed methanotrophic syntrophic associations may also be present. Subsequent enrichment cultures from microcosms yielded communities not predicted by the original diversity studies and showed rates inconsistent with microcosms (≤24.5 µmol d-1), highlighting difficulties in culturing representative thermophilic methanotrophs. Finally, to determine the active methane oxidation processes, we attempted to elucidate metabolic pathways from two enrichment cultures actively oxidizing methane using metatranscriptomics. The most highly expressed genes in both enrichments (methane monooxygenases, methanol dehydrogenases and PqqA precursor peptides) were related to methanotrophs from Methylococcaceae, Methylocystaceae and Methylothermaceae. This is the first example of using metatranscriptomics to investigate methanotrophs from geothermal environments and gives insight into the metabolic pathways involved in thermophilic methanotrophy.

A metatranscriptomic analysis of geothermal hot springs reveals diverse RNA viruses including the phylum Lenarviricota.

Little is known about the diversity of RNA viruses in geothermal systems. We generated total RNA sequencing data from two hot springs in Kuirau Park, Rotorua, New Zealand. In one data set, from a 71.8 °C pool, we observed a microbial community that was 98.5% archaea. The second data set, representing a cooler 36.8 °C geothermal hot spring, had a more diverse microbial profile: 58% bacteria, 34.5% eukaryotes and 7.5% archaea. Within this latter pool, we detected sequences likely representing 23 RNA viruses from the families Astroviridae, Tombusviridae, Polycipiviridae, Discistroviridae, Partitiviridae, and Mitoviridae, as well as from unclassified clades of the orders Tolivirales, Picornavirales, and Ghabrivirales. Most viruses had uncertain host associations. Of particular note, we identified four novel RNA viruses from the phylum Lenarviricota, commonly associated with bacteria and fungi, that occupied a divergent phylogenetic position within unclassified clades and may represent an ancient order-level taxon of unknown host association.

Inference and reconstruction of the heimdallarchaeial ancestry of eukaryotes.

In the ongoing debates about eukaryogenesis-the series of evolutionary events leading to the emergence of the eukaryotic cell from prokaryotic ancestors-members of the Asgard archaea play a key part as the closest archaeal relatives of eukaryotes1. However, the nature and phylogenetic identity of the last common ancestor of Asgard archaea and eukaryotes remain unresolved2-4. Here we analyse distinct phylogenetic marker datasets of an expanded genomic sampling of Asgard archaea and evaluate competing evolutionary scenarios using state-of-the-art phylogenomic approaches. We find that eukaryotes are placed, with high confidence, as a well-nested clade within Asgard archaea and as a sister lineage to Hodarchaeales, a newly proposed order within Heimdallarchaeia. Using sophisticated gene tree and species tree reconciliation approaches, we show that analogous to the evolution of eukaryotic genomes, genome evolution in Asgard archaea involved significantly more gene duplication and fewer gene loss events compared with other archaea. Finally, we infer that the last common ancestor of Asgard archaea was probably a thermophilic chemolithotroph and that the lineage from which eukaryotes evolved adapted to mesophilic conditions and acquired the genetic potential to support a heterotrophic lifestyle. Our work provides key insights into the prokaryote-to-eukaryote transition and a platform for better understanding the emergence of cellular complexity in eukaryotic cells.

Temporal dynamics of geothermal microbial communities in Aotearoa-New Zealand.

Microbial biogeography studies, in particular for geothermal-associated habitats, have focused on spatial patterns and/or individual sites, which have limited ability to describe the dynamics of ecosystem behaviour. Here, we report the first comprehensive temporal study of bacterial and archaeal communities from an extensive range of geothermal features in Aotearoa-New Zealand. One hundred and fifteen water column samples from 31 geothermal ecosystems were taken over a 34-month period to ascertain microbial community stability (control sites), community response to both natural and anthropogenic disturbances in the local environment (disturbed sites) and temporal variation in spring diversity across different pH values (pH 3, 5, 7, 9) all at a similar temperature of 60-70°C (pH sites). Identical methodologies were employed to measure microbial diversity via 16S rRNA gene amplicon sequencing, along with 44 physicochemical parameters from each feature, to ensure confidence in comparing samples across timeframes. Our results indicated temperature and associated groundwater physicochemistry were the most likely parameters to vary stochastically in these geothermal features, with community abundances rather than composition more readily affected by a changing environment. However, variation in pH (pH ±1) had a more significant effect on community structure than temperature (±20°C), with alpha diversity failing to adequately measure temporal microbial disparity in geothermal features outside of circumneutral conditions. While a substantial physicochemical disturbance was required to shift community structures at the phylum level, geothermal ecosystems were resilient at this broad taxonomic rank and returned to a pre-disturbed state if environmental conditions re-established. These findings highlight the diverse controls between different microbial communities within the same habitat-type, expanding our understanding of temporal dynamics in extreme ecosystems.

Description of two cultivated and two uncultivated new Salinibacter species, one named following the rules of the bacteriological code: Salinibacter grassmerensis sp. nov.; and three named following the rules of the SeqCode: Salinibacter pepae sp. nov., Salinibacter abyssi sp. nov., and Salinibacter pampae sp. nov.

Current -omics methods allow the collection of a large amount of information that helps in describing the microbial diversity in nature. Here, and as a result of a culturomic approach that rendered the collection of thousands of isolates from 5 different hypersaline sites (in Spain, USA and New Zealand), we obtained 21 strains that represent two new Salinibacter species. For these species we propose the names Salinibacter pepae sp. nov. and Salinibacter grassmerensis sp. nov. (showing average nucleotide identity (ANI) values < 95.09% and 87.08% with Sal. ruber M31T, respectively). Metabolomics revealed species-specific discriminative profiles. Sal. ruber strains were distinguished by a higher percentage of polyunsaturated fatty acids and specific N-functionalized fatty acids; and Sal. altiplanensis was distinguished by an increased number of glycosylated molecules. Based on sequence characteristics and inferred phenotype of metagenome-assembled genomes (MAGs), we describe two new members of the genus Salinibacter. These species dominated in different sites and always coexisted with Sal. ruber and Sal. pepae. Based on the MAGs from three Argentinian lakes in the Pampa region of Argentina and the MAG of the Romanian lake Fara Fund, we describe the species Salinibacter pampae sp. nov. and Salinibacter abyssi sp. nov. respectively (showing ANI values 90.94% and 91.48% with Sal. ruber M31T, respectively). Sal. grassmerensis sp. nov. name was formed according to the rules of the International Code for Nomenclature of Prokaryotes (ICNP), and Sal. pepae, Sal. pampae sp. nov. and Sal. abyssi sp. nov. are proposed following the rules of the newly published Code of Nomenclature of Prokaryotes Described from Sequence Data (SeqCode). This work constitutes an example on how classification under ICNP and SeqCode can coexist, and how the official naming a cultivated organism for which the deposit in public repositories is difficult finds an intermediate solution.

Machine learning at the edge for AI-enabled multiplexed pathogen detection.

Multiplexed detection of biomarkers in real-time is crucial for sensitive and accurate diagnosis at the point of use. This scenario poses tremendous challenges for detection and identification of signals of varying shape and quality at the edge of the signal-to-noise limit. Here, we demonstrate a robust target identification scheme that utilizes a Deep Neural Network (DNN) for multiplex detection of single particles and molecular biomarkers. The model combines fast wavelet particle detection with Short-Time Fourier Transform analysis, followed by DNN identification on an AI-specific edge device (Google Coral Dev board). The approach is validated using multi-spot optical excitation of Klebsiella Pneumoniae bacterial nucleic acids flowing through an optofluidic waveguide chip that produces fluorescence signals of varying amplitude, duration, and quality. Amplification-free 3× multiplexing in real-time is demonstrated with excellent specificity, sensitivity, and a classification accuracy of 99.8%. These results show that a minimalistic DNN design optimized for mobile devices provides a robust framework for accurate pathogen detection using compact, low-cost diagnostic devices.

Draft Genome Sequence of Venenivibrio stagnispumantis CP.B2T, Isolated from Champagne Pool, Waiotapu, Aotearoa-New Zealand.

Venenivibrio stagnispumantis strain CP.B2T is a thermophilic, chemolithoautotrophic bacterium from the family Hydrogenothermaceae (phylum Aquificota), isolated from Champagne Pool in the Waiotapu geothermal field, Aotearoa-New Zealand. The genome consists of 1.73 Mbp in 451 contigs with a 30.8 mol% G+C content.

An essential role for tungsten in the ecology and evolution of a previously uncultivated lineage of anaerobic, thermophilic Archaea.

Trace metals have been an important ingredient for life throughout Earth's history. Here, we describe the genome-guided cultivation of a member of the elusive archaeal lineage Caldarchaeales (syn. Aigarchaeota), Wolframiiraptor gerlachensis, and its growth dependence on tungsten. A metagenome-assembled genome (MAG) of W. gerlachensis encodes putative tungsten membrane transport systems, as well as pathways for anaerobic oxidation of sugars probably mediated by tungsten-dependent ferredoxin oxidoreductases that are expressed during growth. Catalyzed reporter deposition-fluorescence in-situ hybridization (CARD-FISH) and nanoscale secondary ion mass spectrometry (nanoSIMS) show that W. gerlachensis preferentially assimilates xylose. Phylogenetic analyses of 78 high-quality Wolframiiraptoraceae MAGs from terrestrial and marine hydrothermal systems suggest that tungsten-associated enzymes were present in the last common ancestor of extant Wolframiiraptoraceae. Our observations imply a crucial role for tungsten-dependent metabolism in the origin and evolution of this lineage, and hint at a relic metabolic dependence on this trace metal in early anaerobic thermophiles.

Unique Geothermal Chemistry Shapes Microbial Communities on Mt. Erebus, Antarctica.

Mt. Erebus, Antarctica, is the world's southernmost active volcano and is unique in its isolation from other major active volcanic systems and its distinctive geothermal systems. Using 16S rRNA gene amplicon sequencing and physicochemical analyses, we compared samples collected at two contrasting high-temperature (50°C-65°C) sites on Mt. Erebus: Tramway Ridge, a weather-protected high biomass site, and Western Crater, an extremely exposed low biomass site. Samples were collected along three thermal gradients, one from Western Crater and two within Tramway Ridge, which allowed an examination of the heterogeneity present at Tramway Ridge. We found distinct soil compositions between the two sites, and to a lesser extent within Tramway Ridge, correlated with disparate microbial communities. Notably, pH, not temperature, showed the strongest correlation with these differences. The abundance profiles of several microbial groups were different between the two sites; class Nitrososphaeria amplicon sequence variants (ASVs) dominated the community profiles at Tramway Ridge, whereas Acidobacteriotal ASVs were only found at Western Crater. A co-occurrence network, paired with physicochemical analyses, allowed for finer scale analysis of parameters correlated with differential abundance profiles, with various parameters (total carbon, total nitrogen, soil moisture, soil conductivity, sulfur, phosphorous, and iron) showing significant correlations. ASVs assigned to Chloroflexi classes Ktedonobacteria and Chloroflexia were detected at both sites. Based on the known metabolic capabilities of previously studied members of these groups, we predict that chemolithotrophy is a common strategy in this system. These analyses highlight the importance of conducting broader-scale metagenomics and cultivation efforts at Mt. Erebus to better understand this unique environment.

Fast custom wavelet analysis technique for single molecule detection and identification.

Many sensors operate by detecting and identifying individual events in a time-dependent signal which is challenging if signals are weak and background noise is present. We introduce a powerful, fast, and robust signal analysis technique based on a massively parallel continuous wavelet transform (CWT) algorithm. The superiority of this approach is demonstrated with fluorescence signals from a chip-based, optofluidic single particle sensor. The technique is more accurate than simple peak-finding algorithms and several orders of magnitude faster than existing CWT methods, allowing for real-time data analysis during sensing for the first time. Performance is further increased by applying a custom wavelet to multi-peak signals as demonstrated using amplification-free detection of single bacterial DNAs. A 4x increase in detection rate, a 6x improved error rate, and the ability for extraction of experimental parameters are demonstrated. This cluster-based CWT analysis will enable high-performance, real-time sensing when signal-to-noise is hardware limited, for instance with low-cost sensors in point of care environments.

Optical trapping assisted label-free and amplification-free detection of SARS-CoV-2 RNAs with an optofluidic nanopore sensor.

Ultrasensitive, versatile sensors for molecular biomarkers are a critical component of disease diagnostics and personalized medicine as the COVID-19 pandemic has revealed in dramatic fashion. Integrated electrical nanopore sensors can fill this need via label-free, direct detection of individual biomolecules, but a fully functional device for clinical sample analysis has yet to be developed. Here, we report amplification-free detection of SARS-CoV-2 RNAs with single molecule sensitivity from clinical nasopharyngeal swab samples on an electro-optofluidic chip. The device relies on optically assisted delivery of target carrying microbeads to the nanopore for single RNA detection after release. A sensing rate enhancement of over 2,000x with favorable scaling towards lower concentrations is demonstrated. The combination of target specificity, chip-scale integration and rapid detection ensures the practicality of this approach for COVID-19 diagnosis over the entire clinically relevant concentration range from 104-109 copies/mL.

Seasonal hydrologic and geologic forcing drive hot spring geochemistry and microbial biodiversity.

Hot springs integrate hydrologic and geologic processes that vary over short- and long-term time scales. However, the influence of temporal hydrologic and geologic change on hot spring biodiversity is unknown. Here, we coordinated near-weekly, cross-seasonal (~140 days) geochemical and microbial community analyses of three widely studied hot springs with local precipitation data in Yellowstone National Park. One spring ('HFS') exhibited statistically significant, coupled microbial and geochemical variation across seasons that was associated with recent precipitation patterns. Two other spring communities, 'CP' and 'DS', exhibited minimal to no variation across seasons. Variability in the seasonal response of springs is attributed to differences in the timing and extent of aquifer recharge with oxidized near-surface water from precipitation. This influx of oxidized water is associated with changes in community composition, and in particular, the abundances of aerobic sulfide-/sulfur-oxidizers that can acidify waters. During sampling, a new spring formed after a period of heavy precipitation and its successional dynamics were also influenced by surface water recharge. Collectively, these results indicate that changes in short-term hydrology associated with precipitation can impact hot spring geochemistry and microbial biodiversity. These results point to potential susceptibility of certain hot springs and their biodiversity to sustained, longer-term hydrologic changes.

Optofluidic multiplex detection of single SARS-CoV-2 and influenza A antigens using a novel bright fluorescent probe assay.

The urgency for the development of a sensitive, specific, and rapid point-of-care diagnostic test has deepened during the ongoing COVID-19 pandemic. Here, we introduce an ultrasensitive chip-based antigen test with single protein biomarker sensitivity for the differentiated detection of both severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza A antigens in nasopharyngeal swab samples at diagnostically relevant concentrations. The single-antigen assay is enabled by synthesizing a brightly fluorescent reporter probe, which is incorporated into a bead-based solid-phase extraction assay centered on an antibody sandwich protocol for the capture of target antigens. After optimization of the probe release for detection using ultraviolet light, the full assay is validated with both SARS-CoV-2 and influenza A antigens from clinical nasopharyngeal swab samples (PCR-negative spiked with target antigens). Spectrally multiplexed detection of both targets is implemented by multispot excitation on a multimode interference waveguide platform, and detection at 30 ng/mL with single-antigen sensitivity is reported.

Growth on Formic Acid Is Dependent on Intracellular pH Homeostasis for the Thermoacidophilic Methanotroph Methylacidiphilum sp. RTK17.1.

Members of the genus Methylacidiphilum, a clade of metabolically flexible thermoacidophilic methanotrophs from the phylum Verrucomicrobia, can utilize a variety of substrates including methane, methanol, and hydrogen for growth. However, despite sequentially oxidizing methane to carbon dioxide via methanol and formate intermediates, growth on formate as the only source of reducing equivalents (i.e., NADH) has not yet been demonstrated. In many acidophiles, the inability to grow on organic acids has presumed that diffusion of the protonated form (e.g., formic acid) into the cell is accompanied by deprotonation prompting cytosolic acidification, which leads to the denaturation of vital proteins and the collapse of the proton motive force. In this work, we used a combination of biochemical, physiological, chemostat, and transcriptomic approaches to demonstrate that Methylacidiphilum sp. RTK17.1 can utilize formate as a substrate when cells are able to maintain pH homeostasis. Our findings show that Methylacidiphilum sp. RTK17.1 grows optimally with a circumneutral intracellular pH (pH 6.52 ± 0.04) across an extracellular range of pH 1.5-3.0. In batch experiments, formic acid addition resulted in no observable cell growth and cell death due to acidification of the cytosol. Nevertheless, stable growth on formic acid as the only source of energy was demonstrated in continuous chemostat cultures (D = 0.0052 h-1, td = 133 h). During growth on formic acid, biomass yields remained nearly identical to methanol-grown chemostat cultures when normalized per mole electron equivalent. Transcriptome analysis revealed the key genes associated with stress response: methane, methanol, and formate metabolism were differentially expressed in response to growth on formic acid. Collectively, these results show formic acid represents a utilizable source of energy/carbon to the acidophilic methanotrophs within geothermal environments. Findings expand the known metabolic flexibility of verrucomicrobial methanotrophs to include organic acids and provide insight into potential survival strategies used by these species during methane starvation.

Optofluidic Amplification-free Multiplex Detection of Viral Hemorrhagic Fevers.

Infectious disease outbreaks such as Ebola and other Viral Hemorrhagic Fevers (VHF) require low-complexity, specific, and differentiated diagnostics as illustrated by the recent outbreak in the Democratic Republic of Congo. Here, we describe amplification-free spectrally multiplex detection of four different VHF total RNA samples using multi-spot excitation on a multimode interference waveguide platform along with combinatorial fluorescence labeling of target nucleic acids. In these experiments, we observed an average of 8-fold greater fluorescence signal amplitudes for the Ebola total RNA sample compared to three other total RNA samples: Lake Victoria Marburg Virus, Ravn Marburg Virus, and Crimean-Congo Hemorrhagic Fever. We have attributed this amplitude amplification to an increased amount of RNA during synthesis of soluble glycoprotein in infection. This hypothesis is confirmed by single molecule detection of the total RNA sample after heat-activated release from the carrier microbeads. From these experiments, we observed at least a 5.3x higher RNA mass loading on the Ebola carrier microbeads compared to the Lake Victoria Marburg carrier microbeads, which is consistent with the known production of soluble glycoprotein during infection.

Intra-operative anaesthetic management of older patients undergoing liver surgery.

Older patients represent a growing proportion of the general surgical caseload. This includes those undergoing liver resection, with figures rising faster than the rate of population ageing. The physiology of ageing leads to changes in all body systems which may render the provision of safe anaesthesia more challenging than in younger patients. Anaesthesia for liver surgery has specific principles, largely aimed at reducing venous bleeding from the liver, and those related to complex major surgery. This review explores the principles of anaesthesia for liver resection and describes how they may require modification in the older patient. The traditional approach of low central venous pressure anaesthesia in order to reduce bleeding may need to be altered in the presence of a cardiovascular system less able to tolerate hypotension and hypoperfusion. These changes in physiology should also lower the threshold for invasive monitoring. The provision of effective analgesia perioperatively should be tailored to minimise the surgical stress response and opiate use. Careful consideration of general principles of intra-operative care for older patients, such as positioning, drug dosing, avoidance of excessively deep anaesthesia, and maintenance of normothermia are also important given the prolonged, complex nature of liver surgery. This individualised approach, with careful attention to changes in physiology allows liver resections to be undertaken in older patients without increases in mortality.