Krumholzibacteriota and Deltaproteobacteria contain rare genetic potential to liberate carbon from monoaromatic compounds in subsurface coal seams.

Biogenic methane in subsurface coal seam environments is produced by diverse consortia of microbes. Although this methane is useful for global energy security, it remains unclear which microbes can liberate carbon from the coal. Most of this carbon is relatively resistant to biodegradation, as it is contained within aromatic rings. Thus, to explore for coal-degrading taxa in the subsurface, this study reconstructed relevant metagenome-assembled genomes (MAGs) from coal seams by using a key genomic marker for the anaerobic degradation of monoaromatic compounds as a guide: the benzoyl-CoA reductase gene (bcrABCD). Three MAGs were identified with this genetic potential. The first represented a novel taxon from the Krumholzibacteriota phylum, which this study is the first to describe. This Krumholzibacteriota MAG contained a full set of genes for benzoyl-CoA dearomatization, in addition to other genes for anaerobic catabolism of monoaromatics. Analysis of Krumholzibacteriota MAGs from other environments revealed that this genetic potential may be common, and thus, Krumholzibacteriota may be important organisms for the liberation of recalcitrant carbon in a broad range of environments. Moreover, the assembly and characterization of two Syntrophorhabdus aromaticivorans MAGs from different continents and a Syntrophaceae sp. MAG implicate the Deltaproteobacteria class in coal seam monoaromatic degradation. Each of these taxa are potential rate-limiting organisms for subsurface coal-to-methane biodegradation. Their description here provides some understanding of their function within the coal seam microbiome and will help inform future efforts in coal bed methane stimulation, anoxic bioremediation of organic pollutants, and assessments of anoxic, subsurface carbon cycling and emissions.IMPORTANCESubsurface coal seams are highly anoxic, oligotrophic environments, where the main source of carbon is "locked away" within aromatic rings. Despite these challenges, many coal seams accumulate biogenic methane, implying that the coal seam microbiome is "unlocking" this carbon source in situ. For over two decades, researchers have endeavored to understand which organisms perform these processes. This study provides the first descriptions of organisms with this genetic potential from the coal seam environment. Here, we report metagenomic insights into carbon liberation from aromatic molecules and the degradation pathways involved and describe a Krumholzibacteriota, two Syntrophorhabdus aromaticivorans, and a Syntrophaceae MAG that contain this genetic potential. This is also the first time that the Krumholzibacteriota phylum has been implicated in anaerobic dearomatization of aromatic hydrocarbons. This potential is identified here in numerous MAGs from other terrestrial and marine subsurface habitats, implicating the Krumholzibacteriota in carbon-cycling processes across a broad range of environments.

Anthropogenic petroleum signatures and biodegradation in subantarctic Macquarie Island soils.

Special Antarctic Blend (SAB) diesel is the main fuel used on Macquarie Island and has been identified as the primary contaminant in several past spill events. This study evaluates the environmental impact of petroleum spills at high latitudes, in the soils of subantarctic Macquarie Island. Soil samples were collected from seven locations, including the "fuel farm" and main powerhouse that have been contaminated by petroleum in the past, and five reference locations, away from station infrastructure and from any obvious signs of contamination. Soils were solvent extracted and analysed using gas chromatography-mass spectrometry. The results show that both contaminated and uncontaminated sites contained a suite of different chain-length hydrocarbons. The more contaminated samples from the fuel farm and main powerhouse contained higher concentrations and a greater range of hydrocarbons that typically indicate numerous spills of varying ages. The hydrocarbon signature of samples collected near the fuel farm and at some of the main powerhouse sites was typical of SAB diesel. However, the hydrocarbon signature at other main powerhouse sites suggest contamination with a heavier fuel with different characteristics, including lower pristane/phytane ratios. Traces of C21-C35 cyclic biomarkers in the spill sites may be derived from additional heavier fuels, and include a signature characteristic of crude oil derived from marine carbonate source rocks. Reference samples had lower concentrations of hydrocarbons, and these were dominated by high molecular weight n-alkanes with an odd-carbon-number predominance, typical of higher-plant derived lipids. Some reference samples also contained geochemical signatures that suggest that they too were contaminated by fuel oil. Variable levels of biodegradation of fuels in soils are consistent with a heterogenous site and a relatively slow rate of biodegradation. The occurrence of fresh spilled fuel overprinting biodegraded fuel from earlier spills is compelling evidence of multiple spills and complex mixing in the environment.

Biogeochemistry of Recently Fossilized Siliceous Hot Spring Sinters from Yellowstone, USA.

Active hot springs are dynamic geobiologically active environments. Heat- and element-enriched fluids form hot spring sinter deposits that are inhabited by microbial and macroscopic eukaryotic communities, but it is unclear how variable heat, fluid circulation, and mineralization within hot spring systems affect the preservation of organic matter in sinters. We present geological, petrographic, and organic geochemical data from fossilized hot spring sinters (<13 Ka) from three distinct hot spring fields of Yellowstone National Park. The aims of this study were to examine the preservation of hydrocarbons and discern whether the hydrocarbons in these samples were derived from in situ communities or transported by hydrothermal fluids. Organic geochemistry reveals the presence of n-alkanes, methylalkanes, hopanes, and other terpanes, and the distribution of methylheptadecanes is compared to published observations of community composition in extant hot springs with similar geochemistry. Unexpectedly, hopanes have a thermally mature signal, and Raman spectroscopy confirms that the kerogen in some samples has nearly reached the oil window, despite never having been buried. Our results suggest that organic matter maturation occurred through below-surface processes in the hotter, deeper parts of the hydrothermal system and that this exogenous material was then transported and emplaced within the sinter.

Methanogenic archaea in subsurface coal seams are biogeographically distinct: an analysis of metagenomically-derived mcrA sequences.

The production of methane as an end-product of organic matter degradation in the absence of other terminal electron acceptors is common, and has often been studied in environments such as animal guts, soils and wetlands due to its potency as a greenhouse gas. To date, however, the study of the biogeographic distribution of methanogens across coal seam environments has been minimal. Here, we show that coal seams are host to a diverse range of methanogens, which are distinctive to each geological basin. Based on comparisons to close relatives from other methanogenic environments, the dominant methanogenic pathway in these basins is hydrogenotrophic, with acetoclastic being a second major pathway in the Surat Basin. Finally, mcrA and 16S rRNA gene primer biases were predominantly seen to affect the detection of Methanocellales, Methanomicrobiales and Methanosarcinales taxa in this study. Subsurface coal methanogenic community distributions and pathways presented here provide insights into important metabolites and bacterial partners for in situ coal biodegradation.

Enhancing rhizoremediation of petroleum hydrocarbons through bioaugmentation with a plant growth-promoting bacterial consortium.

The slow rate of natural attenuation of organic pollutants, together with unwanted environmental impacts of traditional remediation strategies, has necessitated the exploration of plant-microbe systems for enhanced bioremediation applications. The identification of microorganisms capable of promoting rhizoremediation through both plant growth-promoting and hydrocarbon-degrading processes is crucial to the success and adoption of plant-based remediation techniques. In this study, through successive enrichments of soil samples from a historic oil-contaminated site in Wietze, Germany, we isolated a plant growth-promoting and hydrocarbon-degrading bacterial consortium dominated by Alphaproteobacteria. In microcosm experiments involving Medicago sativa L. and the isolated bacterial consortium, we examined the ability of the consortium to enhance rhizoremediation of petroleum hydrocarbons. The inoculation of M. sativa with the consortium resulted in 66% increase in plant biomass, and achieved a 91% reduction in diesel fuel hydrocarbon concentrations in the soil within 60 days. Metagenome analysis led to the identification of genes and taxa putatively involved in these processes. The majority of the coding DNA sequences associated with plant growth promotion and hydrocarbon degradation in this study were affiliated to Acidocella aminolytica and Acidobacterium capsulatum indicating their potential for biotechnological applications in the rhizoremediation of sites contaminated by petroleum-derived organic pollutants.

Diversity and metagenome analysis of a hydrocarbon-degrading bacterial consortium from asphalt lakes located in Wietze, Germany.

The pollution of terrestrial and aquatic environments by petroleum contaminants, especially diesel fuel, is a persistent environmental threat requiring cost-effective and environmentally sensitive remediation approaches. Bioremediation is one such approach, but is dependent on the availability of microorganisms with the necessary metabolic abilities and environmental adaptability. The aim of this study was to examine the microbial community in a petroleum contaminated site, and isolate organisms potentially able to degrade hydrocarbons. Through successive enrichment of soil microorganisms from samples of an historic petroleum contaminated site in Wietze, Germany, we isolated a bacterial consortium using diesel fuel hydrocarbons as sole carbon and energy source. The 16S rRNA gene analysis revealed the dominance of Alphaproteobacteria. We further reconstructed a total of 18 genomes from both the original soil sample and the isolated consortium. The analysis of both the metagenome of the consortium and the reconstructed metagenome-assembled genomes show that the most abundant bacterial genus in the consortium, Acidocella, possess many of the genes required for the degradation of diesel fuel aromatic hydrocarbons, which are often the most toxic component. This can explain why this genus proliferated in all the enrichment cultures. Therefore, this study reveals that the microbial consortium isolated in this study and its dominant genus, Acidocella, could potentially serve as an effective inoculum for the bioremediation of sites polluted with diesel fuel or other organic contaminants.

Dose-response analysis of diesel fuel phytotoxicity on selected plant species.

As an ecotoxicological tool, bioassays are an effective screening tool to eliminate plants sensitive to the contaminant of interest, and thereby reduce the number of plant species requiring further study. We conducted a bioassay analysis of fifteen plant species to determine their tolerance to diesel fuel toxicity. Dose-response analysis revealed that increasing diesel fuel concentrations in the soil generally led to a monotonically decreasing biomass in 13 species (P < 0.001), with EC10 values (±SE) ranging from 0.36 ± 0.18 g/kg to 12.67 ± 2.13 g/kg. On the other hand, hydrocarbons had a statistically significant hormetic influence on Medicago sativa (f = 3.90 ± 1.08; P < 0.01). The EC10 and EC50 values (±SE) from the fitted hormetic model were 15.33 ± 1.47 g/kg and 26.89 ± 2.00 g/kg, respectively. While previous studies have shown M. sativa's tolerance of hydrocarbon toxicity, this is the first attempt to describe diesel fuel-induced hormesis in M. sativa using the Cedergreen-Ritz-Streibig model. This study thus shows that hormesis cannot be ignored in plant toxicology research, and that when present, an appropriate statistical model is necessary to avoid drawing wrong conclusions.

Assessing the Effect of Diesel Fuel on the Seed Viability and Germination of Medicago sativa Using the Event-Time Model.

The remediation of contaminated sites using plant-based techniques has gained increasing attention in recent decades. However, information on the effects of contaminant imbibition on seed viability and germination rates are often lacking in the literature. To this end, our research investigated, by means of an event-time model, the effect of diesel fuel imbibition on the seed viability and germination rate of Medicago sativa, a plant species with great potential for remediation of organic contaminants. The event-time model provided an accurate and biologically relevant method for analysing germination data. Our results reveal that the direct imbibition of diesel fuel by M. sativa seeds for ≤48 h, or their exposure to soil diesel fuel concentrations of 0-10 g/kg diesel fuel, affects their germination rates, as shown by increasing t50 values from 90.6 (±2.78) to 114.2 (±2.67) hours, without significantly affecting seed viability. On the other hand, diesel fuel imbibition of longer duration, or the exposure of M. sativa seeds to ≥20 g/kg diesel fuel-contaminated soils, leads to no further effect on time to seed emergence. Instead, these conditions compromise seed viability, resulting in a decrease in the proportion of germinated seeds from 0.91 (±0.03) in 10 g/kg diesel fuel contaminated soil to 0.84 (±0.04) and 0.70 (±0.05) in 20 and 30 g/kg diesel fuel-contaminated soils, respectively. The fact that low concentrations of diesel fuel and 0-48 h of direct imbibition delayed seed emergence without adversely affecting the percentage of viable seeds suggests that this inhibitory effect on germination at low diesel fuel exposure could be attributed more to physical constraints rather than biological damage on the seeds. The models used in this study provide an accurate and biologically relevant method for the analyses of germination data. This is vital since expensive germination experiments, be it in the field of toxicology or agriculture, deserve to be accurately analysed.

Mars Rover Techniques and Lower/Middle Cambrian Microbialites from South Australia: Construction, Biofacies, and Biogeochemistry.

The Perseverance rover (Mars 2020) is equipped with an instrumental and analytical payload capable of identifying a broad range of organic molecules in geological samples. To determine the efficacy of these analytical techniques in recognizing important ecological and environmental signals in the rock record, this study utilized analogous equipment, including gas chromatography/mass spectrometry, Raman spectroscopy, X-ray fluorescence (XRF), Fourier transform infrared spectroscopy, along with macroscopic and petrographic observations, to examine early-middle Cambrian microbialites from the Arrowie Basin, South Australia. Morphological and petrographic observations of these carbonate successions reveal evidence of hypersaline-restricted environments. Microbialites have undergone moderate diagenesis, as supported by XRF data that show mineral assemblages, including celestine and the illitization of smectite. Raman spectral data, carbon preference indices of ∼1, and the methylphenanthrene index place the samples in the prehnite/pumpellyite metamorphic facies. Pristane and phytane are the only biomarkers that were detected in the least thermally mature samples. This research demonstrates a multitechnique approach that can yield significant geological, depositional, paleobiological, and diagenetic information that has important implications for planning future astrobiological exploration.

Biomolecules from Fossilized Hot Spring Sinters: Implications for the Search for Life on Mars.

Hot spring environments are commonly dominated by silica sinters that precipitate by the rapid cooling of silica-saturated fluids and the activity of microbial communities. However, the potential for preservation of organic traces of life in silica sinters back through time is not well understood. This is important for the exploration of early life on Earth and possibly Mars. Most previous studies have focused on physical preservation in samples <900 years old, with only a few focused on organic biomarkers. In this study, we investigate the organic geochemistry of hot spring samples from El Tatio, Chile and the Taupo Volcanic Zone, with ages varying from modern to ∼9.4 ka. Results show that all samples contain opaline silica and contain hydrocarbons that are indicative of a cyanobacterial origin. A ∼3 ka recrystallized, quartz-bearing sample also contains traces of cyanobacterial biomarkers. No aromatic compounds were detected in a ∼9.4 ka opal-A sample or in a modern sinter breccia sample. All other samples contain naphthalene, with one sample also containing other polyaromatic hydrocarbons. These aromatic hydrocarbons have a thermally mature distribution that is perhaps reflective of geothermal fluids migrating from deep, rather than surface, reservoirs. These data show that hot spring sinters can preserve biomolecules from the local microbial community, and that crystallinity rather than age may be the determining factor in their preservation. This research provides support for the exploration for biomolecules in opaline silica deposits on Mars.

Ethanol-blended petroleum fuels: implications of co-solvency for phytotechnologies.

In recent decades, there has been increasing interest in the use of ethanol-blended fuels as alternatives to unblended fossil fuels. These initiatives are targeted at combating CO2 and particulate matter emissions, as these oxygenates leave behind a lesser carbon footprint. Noble as it may appear, this innovation is not without attendant ugly consequences. One major implication is the effect of co-solvency on the applicability of various forms of phytotechnologies for contaminant removal. By means of gas chromatography-mass spectrometry, this research investigated the effect of diesel fuel ethanol addition on the leaching potentials of petroleum hydrocarbons. Since phytoremediation of hydrocarbons depends largely on rhizodegradation of contaminants by the root-associated microbiome, the leaching of petroleum hydrocarbons beyond the rooting zones of plants may limit the effectiveness of this process as a reclamation strategy for ethanol-blended fuel spills. The analyses presented in this paper highlight the need for energy scientists to carefully consider the environmental impacts of ethanol-blended innovations holistically.

Characterization of organophosphatic brachiopod shells: spectroscopic assessment of collagen matrix and biomineral components.

The shells of linguloid brachiopods such as Lingula and Discinisca are inorganic-organic nanocomposites with a mineral phase of calcium phosphate (Ca-phosphate). Collagen, the main extracellular matrix in Ca-phosphatic vertebrate skeletons, has not previously been clearly resolved at the molecular level in organophosphatic brachiopods. Here, modern and recently-alive linguliform brachiopod shells of Lingula and Discinisca have been studied by microRaman spectroscopy, Fourier transform infrared spectroscopy, field emission gun scanning electron microscopy, and thermal gravimetric analysis. For the first time, biomineralized collagen matrix and Ca-phosphate components were simultaneously identified, showing that the collagen matrix is an important moiety in organophosphatic brachiopod shells, in addition to prevalent chitin. Stabilized nanosized apatitic biominerals (up to ∼50 nm) permeate the framework of organic fibrils. There is a ∼2.5-fold higher wt% of carbonate (CO3 2-) in Lingula versus Discinisca shells. Both microRaman spectroscopy and infrared spectra show transient amorphous Ca-phosphate and octacalcium phosphate components. For the first time, trivalent moieties at ∼1660 cm-1 and divalent moieties at ∼1690 cm-1 in the amide I spectral region were identified. These are related to collagen cross-links that are abundant in mineralized tissues, and could be important features in the biostructural and mechanical properties of Ca-phosphate shell biominerals. This work provides a critical new understanding of organophosphatic brachiopod shells, which are some of the earliest examples of biomineralization in still-living animals that appeared in the Cambrian radiation.

Hydrocarbon biomarkers preserved in carbonate veins of potentially Paleoproterozoic age, and implications for the early biosphere.

Research on the early rise of oxygenic photosynthesis and eukaryotes has recently encountered a major pitfall, as some hopane and sterane biomarkers reported in Archaean rocks are the results of contamination. Following an extensive petrological framework in the Pilbara Craton, Western Australia, oil-bearing fluid inclusions and solid bitumens were identified in replacement and hydrothermal carbonate veins cross-cutting Archaean metasedimentary rocks. The 2.55-2.63 billion years old metasedimentary rocks were found to be depleted of indigenous biomarkers. Here we show novel biomarker results from the solvent extraction of the carbonate veins. Volcanic rock blanks, outside rinses, and instrumental blanks showed no biomarkers, and the surrounding rocks were metamorphosed to a sufficiently high extent to not yield any biomarkers, but the biomarkers found in the veins are most likely indigenous. Biomarkers detected include C21-22 ααα- and αßß-steranes (pregnanes), C27-29 αßß-steranes, C19-26 tricyclic terpanes, C29-30,34 αß-hopanes, C30 17α-diahopane, and trisnorhopanes, which are in the range 2-180 pg/g. The extracted organic matter is highly mature, based on the biomarker configurations and calculated vitrinite reflectance that ranges from 2.4-3.0 (methylphenanthrene index), 1.4-1.9 (methyladamantane index), and 1.4-2.3 (methyldiamantane index). As the biomarkers are highly mature and the biomarker assemblages have a distinctive pattern to each vein type the likelihood of sample contamination by recent, less mature, biomarkers from a different assemblage is unlikely. The detection of steranes suggests that molecular oxygen was available when the veins were formed, possibly between 2.2 and 1.8 billion years ago, but no evidence for oxygenic photosynthesis in the form of cyanobacterial biomarkers has been found. Carbonate minerals that seem to better preserve biomarkers, such as concretions or veins, show the growing importance of new and exciting opportunities to seek biomarkers in the early Earth rock record, and potentially on other planets. Our results demonstrate for that first time that biomarkers can be found in veins cutting through highly metamorphosed Archaean rocks, and gives an insight into ancient environments.

The influence of vegetation and soil properties on springtail communities in a diesel-contaminated soil.

Soil health is important for the functioning of all terrestrial ecosystems, but may be impacted by contamination. Soil contamination may in turn necessitate rehabilitation and remediation works, but many of the techniques currently used cause physical disturbance to the soil structure, which may in itself affect soil assemblages. An understanding of the relative influence of these two types of disturbance on soil biota is needed to inform in situ remediation activities. Subantarctic Macquarie Island provides an ideal location to study these interactions because soil biodiversity is naturally low and a number of diesel spills have undergone active in situ remediation in recent years. In this study, soil cores were collected in triplicate from 21 locations. Springtails were extracted and identified to genus/species level. Total petroleum hydrocarbon (TPH) concentrations were measured at the surface and at 0.5m depth at each site, as was vegetation coverage and a range of soil properties. The relationships between these data were examined using distance-based linear models. Together, all environmental variables (vegetation and soil properties) explained a total of 76% of the variation in springtail assemblages. Soil properties alone accounted for 52% of the variation in springtail assemblages, of which bulk density was most important followed by soil conductivity and pH. Vegetation cover by the four plant taxa accounted for 34% of variation observed, with Leptinella plumosa and Poa foliosa having the greatest influence. Surface and underlying TPH concentration did not have a significant effect on springtail assemblages. Overall, factors that can be linked to physical soil disturbance had greater influence over springtail assemblages than did soil contamination. This finding may influence the selection of the most appropriate contaminant management approach for environmentally sensitive sites.

Predicting the weathering of fuel and oil spills: A diffusion-limited evaporation model.

The majority of the evaporation models currently available in the literature for the prediction of oil spill weathering do not take into account diffusion-limited mass transport and the formation of a concentration gradient in the oil phase. The altered surface concentration of the spill caused by diffusion-limited transport leads to a slower evaporation rate compared to the predictions of diffusion-agnostic evaporation models. The model presented in this study incorporates a diffusive layer in the oil phase and predicts the diffusion-limited evaporation rate. The information required is the composition of the fluid from gas chromatography or alternatively the distillation data. If the density or a single viscosity measurement is available the accuracy of the predictions is higher. Environmental conditions such as water temperature, air pressure and wind velocity are taken into account. The model was tested with synthetic mixtures, petroleum fuels and crude oils with initial viscosities ranging from 2 to 13,000 cSt. The tested temperatures varied from 0 °C to 23.4 °C and wind velocities from 0.3 to 3.8 m/s. The average absolute deviation (AAD) of the diffusion-limited model ranged between 1.62% and 24.87%. In comparison, the AAD of a diffusion-agnostic model ranged between 2.34% and 136.62% against the same tested fluids.

Assessing fuel spill risks in polar waters: Temporal dynamics and behaviour of hydrocarbons from Antarctic diesel, marine gas oil and residual fuel oil.

As part of risk assessment of fuel oil spills in Antarctic and subantarctic waters, this study describes partitioning of hydrocarbons from three fuels (Special Antarctic Blend diesel, SAB; marine gas oil, MGO; and intermediate grade fuel oil, IFO 180) into seawater at 0 and 5°C and subsequent depletion over 7days. Initial total hydrocarbon content (THC) of water accommodated fraction (WAF) in seawater was highest for SAB. Rates of THC loss and proportions in equivalent carbon number fractions differed between fuels and over time. THC was most persistent in IFO 180 WAFs and most rapidly depleted in MGO WAF, with depletion for SAB WAF strongly affected by temperature. Concentration and composition remained proportionate in dilution series over time. This study significantly enhances our understanding of fuel behaviour in Antarctic and subantarctic waters, enabling improved predictions for estimates of sensitivities of marine organisms to toxic contaminants from fuels in the region.

Cyanobacterial Inhabitation on Archean Rock Surfaces in the Pilbara Craton, Western Australia.

High abundances of 7- and 6-monomethylalkanes as well as C17 n-alkane, indicative of cyanobacteria, have been discovered near the surfaces of Archean carbonate rocks of the Fortescue Group in the Pilbara region, Western Australia. The presence of cyanobacterial biomarkers is mostly limited to the surface layer (<1 cm thickness) of the rocks, indicating that the cyanobacteria are an endolithic species. Biomarkers are found in bitumen I (solvent-extracted rock) and also in bitumen II (solvent-extracted decarbonated rock). The abundance of biomarkers is generally the same between both bitumen fractions in the surface layer, which suggests that the cyanobacteria penetrated into the carbonate minerals. Trace amounts of the biomarkers have also diffused into a deeper part of the rocks, but this influence is only seen in bitumen I. This implies that hydrocarbons moved toward the inside of the rock through pores and fissures in the rock fabric. In contrast, hydrocarbons in bitumen II, which mainly come from within the carbonate minerals, are isolated from the hydrocarbon migration from the outside of the rock and may be ancient indigenous organic matter. To the best of our knowledge, this is the first report of the past or modern inhabitation of cyanobacteria on Archean rocks in the Pilbara region for which hydrocarbon biomarker analyses was used.

Reappraisal of hydrocarbon biomarkers in Archean rocks.

Hopanes and steranes found in Archean rocks have been presented as key evidence supporting the early rise of oxygenic photosynthesis and eukaryotes, but the syngeneity of these hydrocarbon biomarkers is controversial. To resolve this debate, we performed a multilaboratory study of new cores from the Pilbara Craton, Australia, that were drilled and sampled using unprecedented hydrocarbon-clean protocols. Hopanes and steranes in rock extracts and hydropyrolysates from these new cores were typically at or below our femtogram detection limit, but when they were detectable, they had total hopane (<37.9 pg per gram of rock) and total sterane (<32.9 pg per gram of rock) concentrations comparable to those measured in blanks and negative control samples. In contrast, hopanes and steranes measured in the exteriors of conventionally drilled and curated rocks of stratigraphic equivalence reach concentrations of 389.5 pg per gram of rock and 1,039 pg per gram of rock, respectively. Polycyclic aromatic hydrocarbons and diamondoids, which exceed blank concentrations, exhibit individual concentrations up to 80 ng per gram of rock in rock extracts and up to 1,000 ng per gram of rock in hydropyrolysates from the ultraclean cores. These results demonstrate that previously studied Archean samples host mixtures of biomarker contaminants and indigenous overmature hydrocarbons. Therefore, existing lipid biomarker evidence cannot be invoked to support the emergence of oxygenic photosynthesis and eukaryotes by ∼ 2.7 billion years ago. Although suitable Proterozoic rocks exist, no currently known Archean strata lie within the appropriate thermal maturity window for syngenetic hydrocarbon biomarker preservation, so future exploration for Archean biomarkers should screen for rocks with milder thermal histories.