The use of anaerobic digestion (AD) wastes as nutrient sources for cyanobacterial cultivation offers a sustainable path to closed-loop bioproduction. This study investigated the potential of liquid and solid digestates from a Candidatus Sodalinema alkaliphilum consortium to support community growth under alkaline conditions. While the AD waste provided sufficient nutrients for growth, the culture crashed by the third cultivation cycle, even after dilution of the digestate to mitigate potential inhibitory factors. Microbial community and metabolite analyses suggested that toxic compounds, including 2,4-di-tert-butylphenol (DTBP), likely played a role in the culture crashes, while Roseinatronobacter and other bacteria or archaea from the digestate were unlikely to be direct causes. This study highlights the potential challenges of using AD waste for the cultivation of cyanobacteria in a closed-loop system and emphasizes the need for further investigation into the mechanisms driving culture crashes to enhance the sustainability of this biotechnological approach.

Approximately 3.7 billion years ago, microbial life may have emerged in phosphate-rich salty ponds. Surprisingly, analogs of these environments are present in alkaline lake systems, recognized as highly productive biological ecosystems. In this study, we investigate the microbial ecology of two Canadian soda lake sediment systems characterized by naturally high phosphate levels. Using a comprehensive approach involving geochemistry, metagenomics, and amplicon sequencing, we discovered that groundwater infiltration into Lake Goodenough sediments supported stratified layers of microbial metabolisms fueled by decaying mats. Effective degradation of microbial mats resulted in unexpectedly low net productivity. Evaporation of water from Last Chance Lake and its sediments led to saturation of brines and a habitat dominated by inorganic precipitation reactions, with low productivity, low organic matter turnover and little biological uptake of phosphorus, leading to high phosphate concentrations. Highly alkaline brines were found to be dominated by potentially dormant spore-forming bacteria. These saturated brines also hosted potential symbioses between Halobacteria and Nanoarchaeaota, as well as Lokiarchaea and bacterial sulfate reducers. Metagenome-assembled genomes of Nanoarchaeaota lacked strategies for coping with salty brines and were minimal for Lokiarchaea. Our research highlights that modern analogs for origin-of-life conditions might be better represented by soda lakes with low phosphate concentrations. Thus, highly alkaline brine environments could be too extreme to support origin of life scenarios. These findings shed light on the complex interplay of microbial life in extreme environments and contribute to our understanding of early Earth environments.

Endospore distributions in marine sediments are influenced by geological conduits providing routes for sub-surface to surface microbial dispersal. To examine this phenomenon in more detail, endospore abundance was determined by quantifying dipicolinic acid (DPA) in 16 deep sea sediment cores from hydrocarbon prospective areas in the NW Atlantic Ocean. DPA concentrations were compared with measurements of over 250 different gaseous and liquid hydrocarbon compounds used to assess for the presence of thermogenic hydrocarbons. This revealed significantly higher levels of endospores at hydrocarbon seep sites than at most hydrocarbon-negative sites. In one exceptional case, a hydrocarbon-negative core in close proximity to adjacent thermogenic seep sites exhibited high endospore abundance, indicating either deposition of endospores emitted from the subsurface via nearby seepage, or alternatively a historical thermogenic hydrocarbon seep where hydrocarbons can no longer be detected by standard geochemistry approaches. This work expands the application of DPA as a proxy for quantifying dormant endospore biogeography of both recent and paleoenvironmental hydrocarbon seep sites.

Introduction Shale oil reservoirs are hypothesized to be sterile due to the extremely high temperature, pressure and salinity within these formations (Evans et al. 2018). High concentrations of toxic metals also pose challenges that demand specific microbial adaptions (Boyd and Barkay 2012, White and Gadd 1998, Ben Fekih et al. 2018). While some microorganisms are introduced into and are selected for within shale formations during hydraulic fracturing, the possibility that certain microorganisms are pre-existing inhabitants of these formations is less clear. Here, we followed the microbial diversity of input and output fluids injected into a Montney formation shale reservoir to assess the distribution and transport of microbial populations during hydraulic fracturing. Enrichment cultures distinguished various metabolisms in the microbial populations found in different sample types, and adaptations allowing them to colonize such niches. Material and methods Fracturing fluid, drilling muds (3302 m, 3350 m and 3400 m depths), shale cuttings (rinsed from the drillings muds), shale core plugs and produced water samples (12-month period) were sampled from a Montney shale oil reservoir. Microbial community compositions were analyzed by amplicon sequencing. Metal content was analyzed by inductively coupled plasma-mass spectrophotometry. High salinity enrichments at 90°C of the drilling muds or rinsed shale samples were set up in triplicate and amended with glucose and guar gum (a mannose/galactose-based polymer used during hydraulic fracturing). Sugars were measured through spectrophotometric assays. Metagenomic analyses were performed to assess microbial gene content. Results/Discussion Provenance of microorganisms from the Montney shale formation Provenance of microorganisms from the Montney shale formation Input fluids (fracturing fluid, drilling muds) were revealed to be the likely source of most of the microbial diversity. However, some microorganisms were only detected in the subsurface samples. ASVs affiliated with Aurantimonas , Caminicella , BRH-c8a (Family Desulfallas ) and Geotoga exhibited occurrence patterns consistent with being derived from subsurface shale formations. Geotoga has only ever been reported from oil reservoirs (Semenova et al. 2020). Analysis of produced water revealed ASVs from these groups increasing in abundance during hydraulic fracturing operations, suggesting selective pressure from oil reservoir conditions (e.g., toxic metal presence, input of saline water, temperature and pressure fluctuations). Incubations set up from drilling muds showed a preference for glucose while incubations of the rinsed shale cuttings showed a microbial preference for guar gum (i.e., mannose production; Fig. 0), reinforcing the presence of different populations being derived from surface and subsurface samples. Adaptations for life in Montney shale Adaptations for life in Montney shale When considering adaptations of microorganisms for environmental conditions found in oil reservoirs, it is relevant to note the presence of toxic metals such as arsenic, cadmium and mercury. Levels of all three metals were found to vary over time within the 28-day shale microbial enrichments and 12-month produced water time course analyses (Suppl. material 1). Metagenomics revealed various genes for the internalization and metabolism of all three metals within the shale microbiome (i.e., arsenate reductases, arsenite transporters, metallothioneins, mercuric reductases). In conclusion, the results of this study suggest that shale reservoirs thus might not be sterile environments, and host microorganisms are able to contend with major perturbations.

Oil spilled in marine environments can settle to the seafloor through aggregation and sedimentation processes. This has been predicted to be especially relevant in the Arctic due to plankton blooms initiated by melting sea ice. These conditions exist in the Kivalliq region in Nunavut, Canada, where elevated shipping traffic has increased the risk of accidental spills. Experimental microcosms combining surface sediment and crude oil were incubated at 4 °C over 21 weeks to evaluate the biodegradation potential of seabed microbiomes. Sediments sampled near the communities of Arviat and Chesterfield Inlet were assessed for biodegradation capabilities by combining hydrocarbon geochemistry with 16S rRNA gene and metagenomic sequencing, revealing decreased microbial diversity but enrichment of oil-degrading taxa. Alkane and aromatic hydrocarbon losses corresponded to detection of genes and genomes that encode enzymes for aerobic biodegradation of these compounds, pointing to the utility of marine microbiome surveys for predicting the fate of oil released into Arctic marine environments.

In the Earth’s deep subsurface life is comprised exclusively of microorganisms, and estimates indicate 12-45% of the global prokaryotic biomass, on the order of 10 29 microbes, is found in subseafloor sediments. Investigating how this enormous microbial biomass is maintained in the extreme habitats below seafloor is critical for understanding the rules of life in the deep biosphere. Furthermore, Earth’s subseafloor habitats often present analog environments detected in other planets such as the recently discovered “ocean worlds”, i.e., planetary bodies in our solar system which consist of large subsurface oceans including Saturn’s moons Titan and Enceladus and Jupiter’s moon Europa. Therefore, investigating life in and beneath Earth’s oceans remains at the forefront of the current astrobiological research endeavors. Despite the inhospitable nature of the subseafloor sedimentary realm, active microbial populations including bacteria capable of transforming into dormant endospores have been demonstrated to inhabit deeply buried anoxic sediments and oil reservoirs, permeable ocean crust, and around hydrothermal vents. These extreme habitats often remain physically connected to the seafloor by unique geological features such as marine cold seeps that transmit hydrocarbon-rich fluids originating in deep sediment layers. It remains unclear how fluid migration in cold seeps influence the composition of the seabed microbiome and if they transport deep subsurface life up to the surface. In this study, we addressed this knowledge gap by analyzing over 180 marine surficial sediments from the Gulf of Mexico and the Monterey Bay to assess whether hydrocarbon fluid migration serves as a mechanism for the dispersal of subsurface extremophiles and their introduction into the seabed via cold seeps. Seafloor samples were collected either by piston coring or ROV-operated push coring and were stored at -20°C upon collection. Presence of hydrocarbons in the piston core sediments wa characterized by gas chromatography mass spectrometry and fluorescence spectroscopy whereas gas seepage was determined in the ROV push cores by visual confirmation of gas bubbles emanating from the seafloor. Sediment microbiome composition was determined by high throughput 16S rRNA gene amplicon sequencing. Metabolic diversity was assessed via a genome-centric metagenomics approach aided by shotgun metagenomic sequencing of selected samples. Additionally, viable bacterial endospore communities were investigated from a subset of over 120 of the above samples by allowing endospore germination using a high-temperature incubation assay followed by amplicon sequencing. While 132 of the piston core sediments contained migrated liquid hydrocarbons, evidence of continuous advective transport of thermogenic alkane gases was observed in 11 sediments. Gas seeps harbored distinct microbial communities featuring bacteria and archaea that are well known inhabitants of deep biosphere sediments. Specifically, 25 distinct sequence variants within the bacterial lineages Atribacterota and Aminicenantia and the archaeal lineage Thermoprofundales occurred in significantly greater relative sequence abundance along with well-known seep-colonizing members of the bacterial genus Sulfurovum , in the gas-positive sediments. Metabolic predictions guided by metagenome-assembled genomes suggested these organisms are anaerobic heterotrophs capable of non-respiratory breakdown of organic matter, likely enabling them to inhabit energy-limited deep subseafloor ecosystems. In addition, eight different lineages of anaerobic bacterial endospores activated by sediment incubation assays were strongly associated with hydrocarbon-bearing sediments. These lineages were most closely related to Clostridiales previously detected in oil reservoirs from around the world. These results cumulatively point to petroleum geofluids as a vector for the advection-assisted upward dispersal of deep biosphere microbes from subsurface to surface environments, shaping the microbiome of cold seep sediments and providing a general mechanism for the maintenance of microbial diversity in the deep sea.

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 local redox stratification and microbial community structure. Cultivation-independent biodiversity surveys of microbial communities can offer a 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 Northwest Atlantic Ocean. Cores extending down to 10 m below seafloor (mbsf) were assessed for gas composition, gas isotopes and liquid hydrocarbons. Over 650 bacterial biodiversity libraries were constructed from different sediment depths at these locations. Sites with strong evidence for the presence of thermogenic or biogenic hydrocarbon seepage were reflected in bacterial population analyses that revealed significant differences between hydrocarbon seep and non-seep locations. Specific bacterial indicators were associated with different sediment depth intervals including Caldatribacteriota and Campilobacterota OTUs observed in high relative sequence abundance at 20-50 cmbsf in hydrocarbon seep sediments. These groups were differentially abundant between sites with thermogenic and biogenic hydrocarbon seepage.

Energy from active serpentinization processes potentially fuelled the origin of life on Earth, thus, if serpentinite-impacted sites facilitate microbial habitability, it is important to understand the source and retention of biological signatures in serpentine rocks. Investigating biological signatures in terrestrial analogues of serpentinite-impacted environments is also essential for interpreting molecular signature preservation on extra-terrestrial bodies. To expand knowledge on the types of biological signatures directly derived from living microorganisms (intact polar lipids, IPLs), and both living and dead microorganisms (core lipids), mass spectrometry analysis was performed on Chimaera serpentinite rocks from Antalya Province, Turkey. Brucite rocks were dominated by IPLs from fungal, eukaryotic origin but in contrast, travertine samples had IPL profiles consistent with a mixed fungal, archaeal and bacterial community. The abundance and diversity of archaeal IPLs was significantly higher in the travertine compared to the brucite, and the abundance of archaeal IPLs inside the travertine rock was highest. Archaeal specific core lipids identified inside the brucite rock were not observed as IPL counterparts, suggesting the presence of a non-viable or fossil archaeal community. Comparing IPL profiles with core lipids can discriminate between living microbial communities, necromass, and fossils to combine as a promising molecular tool for identification and interpretation of bio-signatures in serpentinite-impacted sites. Continuing survey of serpentinization samples on Earth can act as analogue environments and provide valuable insight into microbial habitability on Mars and other planetary objects.

ABSTRACT 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.

Energy from serpentinization processes potentially fuelled the origin of life on Earth. Thus, if serpentinite-impacted sites facilitate microbial habitability, it is important to understand the source and retention of bio-logical signatures in them. Investigating biological signatures in terrestrial serpentinite-impacted environments is also essential for interpreting molecular signature preservation on extraterrestrial bodies. To expand knowledge on the types of biological signatures derived from microorganisms in the serpentinization microbiome, mass spectrometry analysis was performed on Chimaera serpentinite rocks from Antalya Province, Turkey. The presence of diverse types of core and intact glycerol alkyl diether and tetraether lipids in all samples indicates that methanogenic archaea were present, but not in equal abundance in all rock types (carbonate or brucite) or locations (inside and outside the rock). Bacterial derived brGDGTs (branched glycerol dialkyl glycerol tet-raethers) and phospholipid fatty acids were also identified in all samples. Comparing targeted lipid data from this study with other studies and locations reveals consistency across the serpentinization lipidome. Additionally, an untargeted lipidomics method was tested on the intact polar lipid (IPL) extracts, This resulted in detection of a wide range of intact lipids, many requiring further elucidation (e.g., diacyl glycerol IPLs, triacylglycerols, gly-cosphingolipids, sterol esters and cardiolipins), suggesting that there may be diverse bacterial, archaeal and fungal communities in the different rock samples, highlighting the additional complexity of the serpentinization lipidome dark matter.

Recent studies have reported up to 1.9 × 1029 bacterial endospores in the upper kilometre of deep subseafloor marine sediments, however, little is understood about their origin and dispersal. In cold ocean environments, the presence of thermospores (endospores produced by thermophilic bacteria) suggests that distribution is governed by passive migration from warm anoxic sources possibly facilitated by geofluid flow, such as advective hydrocarbon seepage sourced from petroleum deposits deeper in the subsurface. This study assesses this hypothesis by measuring endospore abundance and distribution across 60 sites in Eastern Gulf of Mexico (EGM) sediments using a combination of the endospore biomarker 2,6-pyridine dicarboxylic acid or 'dipicolinic acid' (DPA), sequencing 16S rRNA genes of thermospores germinated in 50°C sediment incubations, petroleum geochemistry in the sediments and acoustic seabed data from sub-bottom profiling. High endospore abundance is associated with geologically active conduit features (mud volcanoes, pockmarks, escarpments and fault systems), consistent with subsurface fluid flow dispersing endospores from deep warm sources up into the cold ocean. Thermospores identified at conduit sites were most closely related to bacteria associated with the deep biosphere habitats including hydrocarbon systems. The high endospore abundance at geological seep features demonstrated here suggests that recalcitrant endospores and their chemical components (such as DPA) can be used in concert with geochemical and geophysical analyses to locate discharging seafloor features. This multiproxy approach can be used to better understand patterns of advective fluid flow in regions with complex geology like the EGM basin.

The deep biosphere is the largest microbial habitat on Earth and features abundant bacterial endospores. Whereas dormancy and survival at theoretical energy minima are hallmarks of microbial physiology in the subsurface, ecological processes such as dispersal and selection in the deep biosphere remain poorly understood. We investigated the biogeography of dispersing bacteria in the deep sea where upward hydrocarbon seepage was confirmed by acoustic imagery and geochemistry. Thermophilic endospores in the permanently cold seabed correlated with underlying seep conduits reveal geofluid-facilitated cell migration pathways originating in deep petroleum-bearing sediments. Endospore genomes highlight adaptations to life in anoxic petroleum systems and bear close resemblance to oil reservoir microbiomes globally. Upon transport out of the subsurface, viable thermophilic endospores reenter the geosphere by sediment burial, enabling germination and environmental selection at depth where new petroleum systems establish. This microbial dispersal loop circulates living biomass in and out of the deep biosphere.

AbstractCold surface sediments host a seedbank of functionally diverse thermophilic bacteria. These thermophiles are present as endospores, which are widely dispersed in aquatic environments. Here, we investigated the functional potential of endospore populations in cold surface sediments heated to 80°C. Microbial production of acetate was observed at 80°C and could be enhanced by supplying additional organic carbon substrates. Comparison of 16S rRNA gene amplicon libraries from 80°C enrichments to sediments heated to lower temperatures (50–70°C) showed that temperature selects for distinct populations of endospore‐forming bacteria. Whereas sulfate‐reducing thermophiles were enriched in 50–70°C incubations, 80°C exceeds their thermal tolerance and selects for hyperthermophilic organotrophic bacteria that are similarly detected in amplicon libraries from sediments heated to 90°C. Genome‐resolved metagenomics revealed novel carbon cycling members of Symbiobacteriales, Thermosediminibacteraceae, Thermanaeromonas and Calditerricola with the genomic potential for the degradation of carbohydrates, sugars, amino acids and nucleotides. Endospores of thermophilic bacteria are deposited on seabed sediments worldwide where they remain dormant as they are buried in the accumulating sediments. Our results suggest that endospore populations could be activated by temperature increases encountered during burial and show the potential for organotrophic metabolic activity contributing to acetate generation in deep hot sediments.

Microbially mediated processes in a given habitat tend to be catalyzed by abundant populations that are ecologically adapted to exploit specific environmental characteristics. Typically, metabolic activities of rare populations are limited but may be stimulated in response to acute environmental stressors. Community responses to sudden changes in temperature and pressure can include suppression and activation of different populations, but these dynamics remain poorly understood. The permanently cold ocean floor hosts countless low-abundance microbes including endospores of thermophilic bacteria. Incubating sediments at high temperature resuscitates viable spores, causing the proliferation of bacterial populations. This presents a tractable system for investigating changes in a microbiome's community structure in response to dramatic environmental perturbations. Incubating permanently cold Arctic fjord sediments at 50°C for 216 h with and without volatile fatty acid amendment provoked major changes in community structure. Germination of thermophilic spores from the sediment rare biosphere was tracked using mass spectrometry-based metabolomics, radiotracer-based sulfate reduction rate measurements, and high-throughput 16S rRNA gene sequencing. Comparing community similarity at different intervals of the incubations showed distinct temporal shifts in microbial populations, depending on organic substrate amendment. Metabolite patterns indicated that amino acids and other sediment-derived organics were decomposed by fermentative Clostridia within the first 12–48 h. This fueled early and late phases of exponential increases in sulfate reduction, highlighting the cross-feeding of volatile fatty acids as electron donors for different sulfate-reducing Desulfotomaculia populations. The succession of germinated endospores triggered by sudden exposure to high temperature and controlled by nutrient availability offers a model for understanding the ecological response of dormant microbial communities following major environmental perturbations.

Summary paragraph The deep biosphere is the largest microbial habitat on Earth and features abundant bacterial endospores 1,2 . Whereas dormancy and survival at theoretical energy minima are hallmarks of subsurface microbial populations 3 , the roles of fundamental ecological processes like dispersal and selection in these environments are poorly understood 4 . 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.

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°C). Metabolites for germination and organic matter degradation (dipicolinic acid and organic acids) revealed both endospore germination and subsequent metabolism at ≥80°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.

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At marine cold seeps, gaseous and liquid hydrocarbons migrate from deep subsurface origins to the sediment-water interface. Cold seep sediments are known to host taxonomically diverse microorganisms, but little is known about their metabolic potential and depth distribution in relation to hydrocarbon and electron acceptor availability. Here we combined geophysical, geochemical, metagenomic and metabolomic measurements to profile microbial activities at a newly discovered cold seep in the deep sea. Metagenomic profiling revealed compositional and functional differentiation between near-surface sediments and deeper subsurface layers. In both sulfate-rich and sulfate-depleted depths, various archaeal and bacterial community members are actively oxidizing thermogenic hydrocarbons anaerobically. Depth distributions of hydrocarbon-oxidizing archaea revealed that they are not necessarily associated with sulfate reduction, which is especially surprising for anaerobic ethane and butane oxidizers. Overall, these findings link subseafloor microbiomes to various biochemical mechanisms for the anaerobic degradation of deeply-sourced thermogenic hydrocarbons.

Endospore-forming bacteria make up an important and numerically significant component of microbial communities in a range of settings including soils, industry, hospitals and marine sediments extending into the deep subsurface. Bacterial endospores are non-reproductive structures that protect DNA and improve cell survival during periods unfavourable for bacterial growth. An important determinant of endospores withstanding extreme environmental conditions is 2,6-pyridine dicarboxylic acid (i.e. dipicolinic acid, or DPA), which contributes heat resistance. This study presents an improved HPLC-fluorescence method for DPA quantification using a single 10-min run with pre-column Tb3+ chelation. Relative to existing DPA quantification methods, specific improvements pertain to sensitivity, detection limit and range, as well as the development of new free DPA and spore-specific DPA proxies. The method distinguishes DPA from intact and recently germinated spores, enabling responses to germinants in natural samples or experiments to be assessed in a new way. DPA-based endospore quantification depends on accurate spore-specific DPA contents, in particular, thermophilic spores are shown to have a higher DPA content, meaning that marine sediments with plentiful thermophilic spores may require spore number estimates to be revisited. This method has a wide range of potential applications for more accurately quantifying bacterial endospores in diverse environmental samples.

Over the last decade, glycerol dialkyl glycerol tetraethers (GDGTs) have become one of the most investigated lipid classes in marine and terrestrial organic geochemical research. GDGTs are microbial membrane core lipids biosynthesized as multiple homolog series of isoprenoid or methyl-branched isomers [isoprenoid glycerol dialkyl glycerol tetraethers (isoGDGTs) and Branched GDGTs (brGDGTs), respectively], whose relative abundance depend on a range of environmental parameters, including temperature. This has led to the development of GDGT-based temperature proxies. A key aspect in the analysis of GDGTs and the further development of their use as environmental proxies is good chromatographic separation of the full range of structural and stereo-isomers, with potential for discovery of novel GDGT variants. Several HPLC methods have been developed to this extent, but partial co-elution of GDGTs remains an issue despite long run times. In this study, we investigate the effects of different types of reverse phase (RP) chromatography on the separation of GDGT isomers. We found that the use of a Kinetex C18-XB column gives good separation of isoGDGT isomers in comparison to the recently developed double column HILIC analysis operated in normal phase (NP) and has a shorter run time. In marine samples, the regularly reported isoprenoid GDGTs separated in a similar way as in NP, however an earlier eluting group was observed to elute with the crenarchaeol isomer used in the TEX86 proxy. In a Swedish peat bog sample, a large range of isoGDGT isomers were observed. We observed a range of brGDGT isomers in several samples often with near baseline separation. Exact identification of all these isomers remained elusive, due to the different mechanism of separation in RP, and the complexity of the brGDGT family. The C18-XB method is rapid and versatile and can be set up on either low-pressure HPLC systems (max 400 bar) with a sample run time of 25 min for brGDGTs and 45 min to include isoGDGTs. On UHPLC-MS systems (>600 bar) the sample run time is reduced to 15 min. Most importantly, the C18-XB method presented here gives unusual separation of both isoprenoid and brGDGTs and could be a useful tool for the further elucidation of the biological sources and environmental factors that play a role in the production of different GDGT isomers.