Genomic stasis over millions of years in subseafloor sediment.

One of the significant challenges in microbiology is to understand the extent and mechanisms of evolution within life beneath the surface of the Earth. The population bottleneck that microbes in deep marine sediment experience implies that mutational and population genetic forces could lead to higher levels of relaxed selection and an increase in pseudogenes. To investigate this hypothesis, a group of Thalassospira strains were isolated from subseafloor sediment that is 3 to 6 million years old, as reported by Orsi and colleagues in 2021. These isolates, representing lineages that have been buried for millions of years, offer an excellent opportunity to study the evolution of life beneath the seafloor over a long period. The existence of closely related strains from environments on the surface of the Earth enabled us to examine the impact of selection within each group. We discovered that isolates from beneath the seafloor show lineage-specific similarities to Thalassospira from the surface world, both in the overall intensity of selection on the genome and in the specific genes affected by mutation. We found no signs of increased relaxed selection or other notable genomic changes in the genomes of the Thalassospira isolates from beneath the seafloor, suggesting that these subseafloor isolates were awakened from a million-year near-stasis. The unique genomic characteristics of each Thalassospira lineage from beneath the seafloor must then reflect genetic changes that surface-inhabiting decendants acquired in the past 3-6 million years. Remarkably, Thalassospira lineages beneath the surface appear to have stably maintained their genomes in the midst of metabolic dormancy and extremely long generation times.

Global diversity of microbial communities in marine sediment.

Microbial life in marine sediment contributes substantially to global biomass and is a crucial component of the Earth system. Subseafloor sediment includes both aerobic and anaerobic microbial ecosystems, which persist on very low fluxes of bioavailable energy over geologic time. However, the taxonomic diversity of the marine sedimentary microbial biome and the spatial distribution of that diversity have been poorly constrained on a global scale. We investigated 299 globally distributed sediment core samples from 40 different sites at depths of 0.1 to 678 m below the seafloor. We obtained ∼47 million 16S ribosomal RNA (rRNA) gene sequences using consistent clean subsampling and experimental procedures, which enabled accurate and unbiased comparison of all samples. Statistical analysis reveals significant correlations between taxonomic composition, sedimentary organic carbon concentration, and presence or absence of dissolved oxygen. Extrapolation with two fitted species-area relationship models indicates taxonomic richness in marine sediment to be 7.85 × 103 to 6.10 × 105 and 3.28 × 104 to 2.46 × 106 amplicon sequence variants for Archaea and Bacteria, respectively. This richness is comparable to the richness in topsoil and the richness in seawater, indicating that Bacteria are more diverse than Archaea in Earth's global biosphere.

Endospore abundance, microbial growth and necromass turnover in deep sub-seafloor sediment.

Two decades of scientific ocean drilling have demonstrated widespread microbial life in deep sub-seafloor sediment, and surprisingly high microbial-cell numbers. Despite the ubiquity of life in the deep biosphere, the large community sizes and the low energy fluxes in this vast buried ecosystem are not yet understood. It is not known whether organisms of the deep biosphere are specifically adapted to extremely low energy fluxes or whether most of the observed cells are in a dormant, spore-like state. Here we apply a new approach--the D:L-amino-acid model--to quantify the distributions and turnover times of living microbial biomass, endospores and microbial necromass, as well as to determine their role in the sub-seafloor carbon budget. The approach combines sensitive analyses of unique bacterial markers (muramic acid and D-amino acids) and the bacterial endospore marker, dipicolinic acid, with racemization dynamics of stereo-isomeric amino acids. Endospores are as abundant as vegetative cells and microbial activity is extremely low, leading to microbial biomass turnover times of hundreds to thousands of years. We infer from model calculations that biomass production is sustained by organic carbon deposited from the surface photosynthetic world millions of years ago and that microbial necromass is recycled over timescales of hundreds of thousands of years.

Relationship of Bacterial Richness to Organic Degradation Rate and Sediment Age in Subseafloor Sediment.

UNLABELLED: Subseafloor sediment hosts a large, taxonomically rich, and metabolically diverse microbial ecosystem. However, the factors that control microbial diversity in subseafloor sediment have rarely been explored. Here, we show that bacterial richness varies with organic degradation rate and sediment age. At three open-ocean sites (in the Bering Sea and equatorial Pacific) and one continental margin site (Indian Ocean), richness decreases exponentially with increasing sediment depth. The rate of decrease in richness with increasing depth varies from site to site. The vertical succession of predominant terminal electron acceptors correlates with abundance-weighted community composition but does not drive the vertical decrease in richness. Vertical patterns of richness at the open-ocean sites closely match organic degradation rates; both properties are highest near the seafloor and decline together as sediment depth increases. This relationship suggests that (i) total catabolic activity and/or electron donor diversity exerts a primary influence on bacterial richness in marine sediment and (ii) many bacterial taxa that are poorly adapted for subseafloor sedimentary conditions are degraded in the geologically young sediment, where respiration rates are high. Richness consistently takes a few hundred thousand years to decline from near-seafloor values to much lower values in deep anoxic subseafloor sediment, regardless of sedimentation rate, predominant terminal electron acceptor, or oceanographic context. IMPORTANCE: Subseafloor sediment provides a wonderful opportunity to investigate the drivers of microbial diversity in communities that may have been isolated for millions of years. Our paper shows the impact of in situ conditions on bacterial community structure in subseafloor sediment. Specifically, it shows that bacterial richness in subseafloor sediment declines exponentially with sediment age, and in parallel with organic-fueled oxidation rate. This result suggests that subseafloor diversity ultimately depends on electron donor diversity and/or total community respiration. This work studied how and why biological richness changes over time in the extraordinary ecosystem of subseafloor sediment.

Global distribution of microbial abundance and biomass in subseafloor sediment.

The global geographic distribution of subseafloor sedimentary microbes and the cause(s) of that distribution are largely unexplored. Here, we show that total microbial cell abundance in subseafloor sediment varies between sites by ca. five orders of magnitude. This variation is strongly correlated with mean sedimentation rate and distance from land. Based on these correlations, we estimate global subseafloor sedimentary microbial abundance to be 2.9⋅10(29) cells [corresponding to 4.1 petagram (Pg) C and ∼0.6% of Earth's total living biomass]. This estimate of subseafloor sedimentary microbial abundance is roughly equal to previous estimates of total microbial abundance in seawater and total microbial abundance in soil. It is much lower than previous estimates of subseafloor sedimentary microbial abundance. In consequence, we estimate Earth's total number of microbes and total living biomass to be, respectively, 50-78% and 10-45% lower than previous estimates.

A quantitative model of water radiolysis and chemical production rates near radionuclide-containing solids.

We present a mathematical model that quantifies the rate of water radiolysis near radionuclide-containing solids. Our model incorporates the radioactivity of the solid along with the energies and attenuation properties for alpha (α), beta (ß), and gamma (γ) radiation to calculate volume normalized dose rate profiles. In the model, these dose rate profiles are then used to calculate radiolytic hydrogen (H2) and hydrogen peroxide (H2O2) production rates as a function of distance from the solid-water interface. It expands on previous water radiolysis models by incorporating planar or cylindrical solid-water interfaces and by explicitly including γ radiation in dose rate calculations. To illustrate our model's utility, we quantify radiolytic H2 and H2O2 production rates surrounding spent nuclear fuel under different conditions (at 20 years and 1000 years of storage, as well as before and after barrier failure). These examples demonstrate the extent to which α, ß and γ radiation contributes to total absorbed dose rate and radiolytic production rates. The different cases also illustrate how H2 and H2O2 yields depend on initial composition, shielding and age of the solid. In this way, the examples demonstrate the importance of including all three types of radiation in a general model of total radiolytic production rates.

Subseafloor sedimentary life in the South Pacific Gyre.

The low-productivity South Pacific Gyre (SPG) is Earth's largest oceanic province. Its sediment accumulates extraordinarily slowly (0.1-1 m per million years). This sediment contains a living community that is characterized by very low biomass and very low metabolic activity. At every depth in cored SPG sediment, mean cell abundances are 3 to 4 orders of magnitude lower than at the same depths in all previously explored subseafloor communities. The net rate of respiration by the subseafloor sedimentary community at each SPG site is 1 to 3 orders of magnitude lower than the rates at previously explored sites. Because of the low respiration rates and the thinness of the sediment, interstitial waters are oxic throughout the sediment column in most of this region. Consequently, the sedimentary community of the SPG is predominantly aerobic, unlike previously explored subseafloor communities. Generation of H(2) by radiolysis of water is a significant electron-donor source for this community. The per-cell respiration rates of this community are about 2 orders of magnitude higher (in oxidation/reduction equivalents) than in previously explored anaerobic subseafloor communities. Respiration rates and cell concentrations in subseafloor sediment throughout almost half of the world ocean may approach those in SPG sediment.

Patterns of Relative Bacterial Richness and Community Composition in Seawater and Marine Sediment Are Robust for Both Operational Taxonomic Units and Amplicon Sequence Variants.

To understand the relative influences of operational taxonomic units (OTUs) and amplicon sequence variants (ASVs) on patterns of marine microbial diversity and community composition, we examined bacterial diversity and community composition of seawater from 12 sites in the North Atlantic Ocean and Canadian Arctic and sediment from two sites in the North Atlantic. For the seawater analyses, we included samples from three to six zones in the water column of each site. For the sediment analyses, we included over 20 sediment horizons at each of two sites. For all samples, we amplified the V4-V5 hypervariable region of the 16S ribosomal RNA (rRNA) gene. We analyzed each sample in two different ways: (i) by clustering its reads into 97%-similar OTUs and (ii) by assigning sequences to unique ASVs. OTU richness is much higher than ASV richness for every sample, but both OTUs and ASVs exhibit similar vertical patterns of relative diversity in both the water column and the sediment. Bacterial richness is highest just below the photic zone in the water column and at the seafloor in the sediment. For both OTUs and ASVs, richness estimates depend on the number of sequences analyzed. Both methods yield broadly similar community compositions for each sample at the taxonomic levels of phyla to families. While the two methods yield different richness values, broad-scale patterns of relative richness and community composition are similar with both methods.

Cryptic metabolisms in anoxic subseafloor sediment.

Microbial gene expression in anoxic subseafloor sediment was recently explored in the Baltic Sea and the Peru Margin. Our analysis of these data reveals diverse transcripts encoding proteins associated with neutralization of reactive oxygen species, including catalase, which may provide an in situ source of oxygen. We also detect transcripts associated with oxidation of iron and sulfur, and with reduction of arsenate, selenate and nitrate. Given limited input of electron acceptors from outside the system, these results suggest that the microbial communities use an unexpectedly diverse variety of electron acceptors. Products of water radiolysis and their interactions with sediment continuously provide diverse electron acceptors and hydrogen. Cryptic microbial utilization of these oxidized substrates and H2 may be an important mechanism for multi-million-year survival under the extreme energy limitation in subseafloor sediment.

Genome Evolution in Bacteria Isolated from Million-Year-Old Subseafloor Sediment.

Beneath the seafloor, microbial life subsists in isolation from the surface world under persistent energy limitation. The nature and extent of genomic evolution in subseafloor microbes have been unknown. Here, we show that the genomes of Thalassospira bacterial populations cultured from million-year-old subseafloor sediments evolve in clonal populations by point mutation, with a relatively low rate of homologous recombination and elevated numbers of pseudogenes. Ratios of nonsynonymous to synonymous substitutions correlate with the accumulation of pseudogenes, consistent with a role for genetic drift in the subseafloor strains but not in type strains of Thalassospira isolated from the surface world. Consistent with this, pangenome analysis reveals that the subseafloor bacterial genomes have a significantly lower number of singleton genes than the type strains, indicating a reduction in recent gene acquisitions. Numerous insertion-deletion events and pseudogenes were present in a flagellar operon of the subseafloor bacteria, indicating that motility is nonessential in these million-year-old subseafloor sediments. This genomic evolution in subseafloor clonal populations coincided with a phenotypic difference: all subseafloor isolates have a lower rate of growth under laboratory conditions than the Thalassospira xiamenensis type strain. Our findings demonstrate that the long-term physical isolation of Thalassospira, in the absence of recombination, has resulted in clonal populations whereby reduced access to novel genetic material from neighbors has resulted in the fixation of new mutations that accumulate in genomes over millions of years. IMPORTANCE The nature and extent of genomic evolution in subseafloor microbial populations subsisting for millions of years below the seafloor are unknown. Subseafloor populations have ultralow metabolic rates that are hypothesized to restrict reproduction and, consequently, the spread of new traits. Our findings demonstrate that genomes of cultivated bacterial strains from the genus Thalassospira isolated from million-year-old abyssal sediment exhibit greatly reduced levels of homologous recombination, elevated numbers of pseudogenes, and genome-wide evidence of relaxed purifying selection. These substitutions and pseudogenes are fixed into the population, suggesting that the genome evolution of these bacteria has been dominated by genetic drift. Thus, reduced recombination, stemming from long-term physical isolation, resulted in small clonal populations of Thalassospira that have accumulated mutations in their genomes over millions of years.

The contribution of water radiolysis to marine sedimentary life.

Water radiolysis continuously produces H2 and oxidized chemicals in wet sediment and rock. Radiolytic H2 has been identified as the primary electron donor (food) for microorganisms in continental aquifers kilometers below Earth's surface. Radiolytic products may also be significant for sustaining life in subseafloor sediment and subsurface environments of other planets. However, the extent to which most subsurface ecosystems rely on radiolytic products has been poorly constrained, due to incomplete understanding of radiolytic chemical yields in natural environments. Here we show that all common marine sediment types catalyse radiolytic H2 production, amplifying yields by up to 27X relative to pure water. In electron equivalents, the global rate of radiolytic H2 production in marine sediment appears to be 1-2% of the global organic flux to the seafloor. However, most organic matter is consumed at or near the seafloor, whereas radiolytic H2 is produced at all sediment depths. Comparison of radiolytic H2 consumption rates to organic oxidation rates suggests that water radiolysis is the principal source of biologically accessible energy for microbial communities in marine sediment older than a few million years. Where water permeates similarly catalytic material on other worlds, life may also be sustained by water radiolysis.

Exploring the abundance, metabolic potential and gene expression of subseafloor Chloroflexi in million-year-old oxic and anoxic abyssal clay.

Chloroflexi are widespread in subsurface environments, and recent studies indicate that they represent a major fraction of the communities in subseafloor sediment. Here, we compare the abundance, diversity, metabolic potential and gene expression of Chloroflexi from three abyssal sediment cores from the western North Atlantic Gyre (water depth >5400 m) covering up to 15 million years of sediment deposition, where Chloroflexi were found to represent major components of the community at all sites. Chloroflexi communities die off in oxic red clay over 10-15 million years, and gene expression was below detection. In contrast, Chloroflexi abundance and gene expression at the anoxic abyssal clay site increase below the seafloor and peak in 2-3 million-year-old sediment, indicating a comparably higher activity. Metatranscriptomes from the anoxic site reveal increased expression of Chloroflexi genes involved in cell wall biogenesis, protein turnover, inorganic ion transport, defense mechanisms and prophages. Phylogenetic analysis shows that these Chloroflexi are closely related to homoacetogenic subseafloor clades and actively transcribe genes involved in sugar fermentations, gluconeogenesis and Wood-Ljungdahl pathway in the subseafloor. Concomitant expression of cell division genes indicates that these putative homoacetogenic Chloroflexi are actively growing in these million-year-old anoxic abyssal sediments.

Atribacteria Reproducing over Millions of Years in the Atlantic Abyssal Subseafloor.

How microbial metabolism is translated into cellular reproduction under energy-limited settings below the seafloor over long timescales is poorly understood. Here, we show that microbial abundance increases an order of magnitude over a 5 million-year-long sequence in anoxic subseafloor clay of the abyssal North Atlantic Ocean. This increase in biomass correlated with an increased number of transcribed protein-encoding genes that included those involved in cytokinesis, demonstrating that active microbial reproduction outpaces cell death in these ancient sediments. Metagenomes, metatranscriptomes, and 16S rRNA gene sequencing all show that the actively reproducing community was dominated by the candidate phylum "Candidatus Atribacteria," which exhibited patterns of gene expression consistent with fermentative, and potentially acetogenic, metabolism. "Ca. Atribacteria" dominated throughout the 8 million-year-old cored sequence, despite the detection limit for gene expression being reached in 5 million-year-old sediments. The subseafloor reproducing "Ca. Atribacteria" also expressed genes encoding a bacterial microcompartment that has potential to assist in secondary fermentation by recycling aldehydes and, thereby, harness additional power to reduce ferredoxin and NAD+ Expression of genes encoding the Rnf complex for generation of chemiosmotic ATP synthesis were also detected from the subseafloor "Ca Atribacteria," as well as the Wood-Ljungdahl pathway that could potentially have an anabolic or catabolic function. The correlation of this metabolism with cytokinesis gene expression and a net increase in biomass over the million-year-old sampled interval indicates that the "Ca Atribacteria" can perform the necessary catabolic and anabolic functions necessary for cellular reproduction, even under energy limitation in millions-of-years-old anoxic sediments.IMPORTANCE The deep subseafloor sedimentary biosphere is one of the largest ecosystems on Earth, where microbes subsist under energy-limited conditions over long timescales. It remains poorly understood how mechanisms of microbial metabolism promote increased fitness in these settings. We discovered that the candidate bacterial phylum "Candidatus Atribacteria" dominated a deep-sea subseafloor ecosystem, where it exhibited increased transcription of genes associated with acetogenic fermentation and reproduction in million-year-old sediment. We attribute its improved fitness after burial in the seabed to its capabilities to derive energy from increasingly oxidized metabolites via a bacterial microcompartment and utilize a potentially reversible Wood-Ljungdahl pathway to help meet anabolic and catabolic requirements for growth. Our findings show that "Ca Atribacteria" can perform all the necessary catabolic and anabolic functions necessary for cellular reproduction, even under energy limitation in anoxic sediments that are millions of years old.

Aerobic microbial life persists in oxic marine sediment as old as 101.5 million years.

Sparse microbial populations persist from seafloor to basement in the slowly accumulating oxic sediment of the oligotrophic South Pacific Gyre (SPG). The physiological status of these communities, including their substrate metabolism, is previously unconstrained. Here we show that diverse aerobic members of communities in SPG sediments (4.3‒101.5 Ma) are capable of readily incorporating carbon and nitrogen substrates and dividing. Most of the 6986 individual cells analyzed with nanometer-scale secondary ion mass spectrometry (NanoSIMS) actively incorporated isotope-labeled substrates. Many cells responded rapidly to incubation conditions, increasing total numbers by 4 orders of magnitude and taking up labeled carbon and nitrogen within 68 days after incubation. The response was generally faster (on average, 3.09 times) for nitrogen incorporation than for carbon incorporation. In contrast, anaerobic microbes were only minimally revived from this oxic sediment. Our results suggest that microbial communities widely distributed in organic-poor abyssal sediment consist mainly of aerobes that retain their metabolic potential under extremely low-energy conditions for up to 101.5 Ma.

Deep microbial proliferation at the basalt interface in 33.5-104 million-year-old oceanic crust.

The upper oceanic crust is mainly composed of basaltic lava that constitutes one of the largest habitable zones on Earth. However, the nature of deep microbial life in oceanic crust remains poorly understood, especially where old cold basaltic rock interacts with seawater beneath sediment. Here we show that microbial cells are densely concentrated in Fe-rich smectite on fracture surfaces and veins in 33.5- and 104-million-year-old (Ma) subseafloor basaltic rock. The Fe-rich smectite is locally enriched in organic carbon. Nanoscale solid characterizations reveal the organic carbon to be microbial cells within the Fe-rich smectite, with cell densities locally exceeding 1010 cells/cm3. Dominance of heterotrophic bacteria indicated by analyses of DNA sequences and lipids supports the importance of organic matter as carbon and energy sources in subseafloor basalt. Given the prominence of basaltic lava on Earth and Mars, microbial life could be habitable where subsurface basaltic rocks interact with liquid water.

Microbial Selection and Survival in Subseafloor Sediment.

Many studies have examined relationships of microorganisms to geochemical zones in subseafloor sediment. However, responses to selective pressure and patterns of community succession with sediment depth have rarely been examined. Here we use 16S rDNA sequencing to examine the succession of microbial communities at sites in the Indian Ocean and the Bering Sea. The sediment ranges in depth from 0.16 to 332 m below seafloor and in age from 660 to 1,300,000 years. The majority of subseafloor taxonomic diversity is present in the shallowest depth sampled. The best predictor of sequence presence or absence in the oldest sediment is relative abundance in the near-seafloor sediment. This relationship suggests that perseverance of specific taxa into deep, old sediment is primarily controlled by the taxonomic abundance that existed when the sediment was near the seafloor. The operational taxonomic units that dominate at depth comprise a subset of the local seafloor community at each site, rather than a grown-in group of geographically widespread subseafloor specialists. At both sites, most taxa classified as abundant decrease in relative frequency with increasing sediment depth and age. Comparison of community composition to cell counts at the Bering Sea site indicates that the rise of the few dominant taxa in the deep subseafloor community does not require net replication, but might simply result from lower mortality relative to competing taxa on the long timescale of community burial.

Influence of 16S rRNA Hypervariable Region on Estimates of Bacterial Diversity and Community Composition in Seawater and Marine Sediment.

To assess the influence of 16S ribosomal RNA (rRNA) tag choice on estimates of microbial diversity and/or community composition in seawater and marine sediment, we examined bacterial diversity and community composition from a site in the Central North Atlantic and a site in the Equatorial Pacific. For each site, we analyzed samples from four zones in the water column, a seafloor sediment sample, and two subseafloor sediment horizons (with stratigraphic ages of 1.5 and 5.5 million years old). We amplified both the V4 and V6 hypervariable regions of the 16S rRNA gene and clustered the sequences into operational taxonomic units (OTUs) of 97% similarity to analyze for diversity and community composition. OTU richness is much higher with the V6 tag than with the V4 tag, and subsequently OTU-level community composition is quite different between the two tags. Vertical patterns of relative diversity are broadly the same for both tags, with maximum taxonomic richness in seafloor sediment and lowest richness in subseafloor sediment at both geographic locations. Genetic dissimilarity between sample locations is also broadly the same for both tags. Community composition is very similar for both tags at the class level, but very different at the level of 97% similar OTUs. Class-level diversity and community composition of water-column samples are very similar at each water depth between the Atlantic and Pacific. However, sediment communities differ greatly from the Atlantic site to the Pacific site. Finally, for relative patterns of diversity and class-level community composition, deep sequencing and shallow sequencing provide similar results.

Subseafloor life and its biogeochemical impacts.

Subseafloor microbial activities are central to Earth's biogeochemical cycles. They control Earth's surface oxidation and major aspects of ocean chemistry. They affect climate on long timescales and play major roles in forming and destroying economic resources. In this review, we evaluate present understanding of subseafloor microbes and their activities, identify research gaps, and recommend approaches to filling those gaps. Our synthesis suggests that chemical diffusion rates and reaction affinities play a primary role in controlling rates of subseafloor activities. Fundamental aspects of subseafloor communities, including features that enable their persistence at low catabolic rates for millions of years, remain unknown.

Archaea dominate oxic subseafloor communities over multimillion-year time scales.

Ammonia-oxidizing archaea (AOA) dominate microbial communities throughout oxic subseafloor sediment deposited over millions of years in the North Atlantic Ocean. Rates of nitrification correlated with the abundance of these dominant AOA populations, whose metabolism is characterized by ammonia oxidation, mixotrophic utilization of organic nitrogen, deamination, and the energetically efficient chemolithoautotrophic hydroxypropionate/hydroxybutyrate carbon fixation cycle. These AOA thus have the potential to couple mixotrophic and chemolithoautotrophic metabolism via mixotrophic deamination of organic nitrogen, followed by oxidation of the regenerated ammonia for additional energy to fuel carbon fixation. This metabolic feature likely reduces energy loss and improves AOA fitness under energy-starved, oxic conditions, thereby allowing them to outcompete other taxa for millions of years.

Radiolytic H2 Production in Martian Environments.

Hydrogen, produced by water radiolysis, has been suggested to support microbial communities on Mars. We quantitatively assess the potential magnitude of radiolytic H2 production in wet martian environments (the ancient surface and the present subsurface) based on the radionuclide compositions of (1) eight proposed Mars 2020 landing sites, and (2) three sites that individually yield the highest or lowest calculated radiolytic H2 production rates on Mars. For the proposed landing sites, calculated H2 production rates vary by a factor of ∼1.6, while the three comparison sites differ by a factor of ∼6. Rates in wet martian sediment and microfractured rock are comparable with rates in terrestrial environments that harbor low concentrations of microbial life (e.g., subseafloor basalt). Calculated H2 production rates for low-porosity (<35%), fine-grained martian sediment (0.12-1.2 nM/year) are mostly higher than rates for South Pacific subseafloor basalt (∼0.02-0.6 nM/year). Production rates in martian high-porosity sediment (>35%) and microfractured (1 µm) hard rock (0.03 to <0.71 nM/year) are generally similar to rates in South Pacific basalt, while yields for larger martian fractures (1 and 10 cm) are one to two orders of magnitude lower (<0.01 nM/year). If minerals or brine that amplify radiolytic H2 production rates are present, H2 yields exceed the calculated rates.