Extreme Energy Limitation Research Articles

Viruses, Vesicles, and other Biological Nanoparticles: The Sub-cellular Biosphere of a Deeply Buried 2km-Deep, 20-Million-Year-Old Coalbed Community

Horizontal gene transfer is an important driver of adaptation and evolution in microorganisms. Transducing biological nanoparticles such as viral particles are believed to be key facilitators of horizontal gene transfer. In deep subseafloor sediments, energy can be highly limiting, supporting only extremely slow metabolisms. In such low-energy, isolated environments where communities may subsist for millions of years, the mechanisms of subsurface microbial adaptation and evolution remain a mystery. Virus particles have been found everywhere that life has been found, including deep subsurface environments. Although microorganisms are abundant and active in the Earth's subsurface, the role of viruses in shaping and influencing these slow-growing communities is only recently starting to be explored. Here, we analyzed the deeply buried microbial community from a lignite coalbed layer 2km below the seafloor offshore Shimokita, Japan (IODP Expedition 337) that had been buried for 20 million years. We harvested cells (>0.2µm) and biological nanoparticles (<0.2µm) from a bioreactor enrichment seeded by lignite core samples. We sequenced DNA from the cells and nanoparticles and subsequently analyzed the metagenomes. Within the nanoparticle metagenome, numerous complete novel virus genomes were reconstructed. Comparison of the virus genomes to the prokaryotic MAGs (metagenome assembled genomes) revealed that many of the virus genomes had been integrated prophage within bacterial genomes, suggesting the potential for virus-host interactions to occur in the deep subseafloor. Additionally, lysogeny may be an important survival mechanism for viruses in deeply buried, low-energy environments. Host genes were found to be packaged by viral particles, demonstrating the potential for specialized and general transduction by viruses. Not only viral particles, but there was also evidence that membrane vesicles and gene transfer agents may participate in transduction in this deep subsurface community. Horizontal gene transfer mediated by biological nanoparticles may be an important mechanism of adaptation for deep subsurface microbial communities and may provide insight into possible evolutionary processes shaping microbial communities in the deep subsurface. These results may also shed some light onto the nature of viral infection in the subsurface, potentially revealing insights about the long-term persistence of life under extreme energy limitation and how viruses may survive this over geological timescales.

Multiple energy sources and metabolic strategies sustain microbial diversity in Antarctic desert soils

Numerous diverse microorganisms reside in the cold desert soils of continental Antarctica, though we lack a holistic understanding of the metabolic processes that sustain them. Here, we profile the composition, capabilities, and activities of the microbial communities in 16 physicochemically diverse mountainous and glacial soils. We assembled 451 metagenome-assembled genomes from 18 microbial phyla and inferred through Bayesian divergence analysis that the dominant lineages present are likely native to Antarctica. In support of earlier findings, metagenomic analysis revealed that the most abundant and prevalent microorganisms are metabolically versatile aerobes that use atmospheric hydrogen to support aerobic respiration and sometimes carbon fixation. Surprisingly, however, hydrogen oxidation in this region was catalyzed primarily by a phylogenetically and structurally distinct enzyme, the group 1l [NiFe]-hydrogenase, encoded by nine bacterial phyla. Through gas chromatography, we provide evidence that both Antarctic soil communities and an axenic Bacteroidota isolate (Hymenobacter roseosalivarius) oxidize atmospheric hydrogen using this enzyme. Based on ex situ rates at environmentally representative temperatures, hydrogen oxidation is theoretically sufficient for soil communities to meet energy requirements and, through metabolic water production, sustain hydration. Diverse carbon monoxide oxidizers and abundant methanotrophs were also active in the soils. We also recovered genomes of microorganisms capable of oxidizing edaphic inorganic nitrogen, sulfur, and iron compounds and harvesting solar energy via microbial rhodopsins and conventional photosystems. Obligately symbiotic bacteria, including Patescibacteria, Chlamydiae, and predatory Bdellovibrionota, were also present. We conclude that microbial diversity in Antarctic soils reflects the coexistence of metabolically flexible mixotrophs with metabolically constrained specialists.

Microbial population dynamics and evolutionary outcomes under extreme energy limitation

Microorganisms commonly inhabit energy-limited ecosystems where cellular maintenance and reproduction is highly constrained. To gain insight into how individuals persist under such conditions, we derived demographic parameters from a collection of 21 heterotrophic bacterial taxa by censusing 100 populations in an effectively closed system for 1,000 d. All but one taxon survived prolonged resource scarcity, yielding estimated times to extinction ranging over four orders of magnitude from 100 to 105 y. Our findings corroborate reports of long-lived bacteria recovered from ancient environmental samples, while providing insight into mechanisms of persistence. As death rates declined over time, lifespan was extended through the scavenging of dead cells. Although reproduction was suppressed in the absence of exogenous resources, populations continued to evolve. Hundreds of mutations were acquired, contributing to genome-wide signatures of purifying selection as well as molecular signals of adaptation. Consistent ecological and evolutionary dynamics indicate that distantly related bacteria respond to energy limitation in a similar and predictable manner, which likely contributes to the stability and robustness of microbial life.

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.

Contribution of Complex I NADH Dehydrogenase to Respiratory Energy Coupling in Glucose-Grown Cultures of Ogataea parapolymorpha

The thermotolerant yeast Ogataea parapolymorpha (formerly Hansenula polymorpha) is an industrially relevant production host that exhibits a fully respiratory sugar metabolism in aerobic batch cultures. NADH-derived electrons can enter its mitochondrial respiratory chain either via a proton-translocating complex I NADH-dehydrogenase or via three putative alternative NADH dehydrogenases. This respiratory entry point affects the amount of ATP produced per NADH/O2 consumed and therefore impacts the maximum yield of biomass and/or cellular products from a given amount of substrate. To investigate the physiological importance of complex I, a wild-type O. parapolymorpha strain and a congenic complex I-deficient mutant were grown on glucose in aerobic batch, chemostat, and retentostat cultures in bioreactors. In batch cultures, the two strains exhibited a fully respiratory metabolism and showed the same growth rates and biomass yields, indicating that, under these conditions, the contribution of NADH oxidation via complex I was negligible. Both strains also exhibited a respiratory metabolism in glucose-limited chemostat cultures, but the complex I-deficient mutant showed considerably reduced biomass yields on substrate and oxygen, consistent with a lower efficiency of respiratory energy coupling. In glucose-limited retentostat cultures at specific growth rates down to ∼0.001 h-1, both O. parapolymorpha strains showed high viability. Maintenance energy requirements at these extremely low growth rates were approximately 3-fold lower than estimated from faster-growing chemostat cultures, indicating a stringent-response-like behavior. Quantitative transcriptome and proteome analyses indicated condition-dependent expression patterns of complex I subunits and of alternative NADH dehydrogenases that were consistent with physiological observations.IMPORTANCE Since popular microbial cell factories have typically not been selected for efficient respiratory energy coupling, their ATP yields from sugar catabolism are often suboptimal. In aerobic industrial processes, suboptimal energy coupling results in reduced product yields on sugar, increased process costs for oxygen transfer, and volumetric productivity limitations due to limitations in gas transfer and cooling. This study provides insights into the contribution of mechanisms of respiratory energy coupling in the yeast cell factory Ogataea parapolymorpha under different growth conditions and provides a basis for rational improvement of energy coupling in yeast cell factories. Analysis of energy metabolism of O. parapolymorpha at extremely low specific growth rates indicated that this yeast reduces its energy requirements for cellular maintenance under extreme energy limitation. Exploration of the mechanisms for this increased energetic efficiency may contribute to an optimization of the performance of industrial processes with slow-growing eukaryotic cell factories.

Sub‐seafloor biogeochemical processes and microbial life in the Baltic Sea

The post-glacial Baltic Sea has experienced extreme changes that are archived today in the deep sediments. IODP Expedition 347 retrieved cores down to 100 m depth and studied the climate history and the deep biosphere. We here review the biogeochemical and microbiological highlights and integrate these with other studies from the Baltic seabed. Cell numbers, endospore abundance and organic matter mineralization rates are extremely high. A 100-fold drop in cell numbers with depth results from a small difference between growth and mortality in the ageing sediment. Evidence for growth derives from a D:L amino acid racemization model, while evidence for mortality derives from the abundance and potential activity of lytic viruses. The deep communities assemble at the bottom of the bioturbated zone from the founding surface community by selection of organisms suited for life under deep sediment conditions. The mean catabolic per-cell rate of microorganisms drops steeply with depth to a life in slow-motion, typical for the deep biosphere. The subsurface life under extreme energy limitation is facilitated by exploitation of recalcitrant substrates, by biochemical protection of nucleic acids and proteins and by repair mechanisms for random mismatches in DNA or damaged amino acids in proteins.

Microbial biomass turnover times and clues to cellular protein repair in energy-limited deep Baltic Sea sediments.

The discovery of active microbial life deeply buried beneath the seafloor has opened important questions: how do microorganisms cope with extreme energy limitation, what is their metabolic activity, and how do they repair damages to essential biomolecules? We used a D:L-amino acid model to calculate microbial biomass turnover times. We used a metagenome and metatranscriptome analysis to investigate the distribution of the gene that encodes Protein-L-iso aspartate(D-aspartate) O-methyltransferase (PCMT), an enzyme which recognizes damaged L-isoapartyl and D-aspartyl residues in proteins and catalyzes their repair. Sediment was retrieved during the Integrated Ocean Drilling Program (IODP) Expedition 347 from Landsort Deep and the Little Belt in the Baltic Sea. The study covers the period from the Baltic Ice Lake ca. 13 000 years ago to the present. Our results provide new knowledge on microbial biomass turnover times and protein repair in relation to different regimes of organic matter input. For the first time, we show that the PCMT gene was widely distributed and expressed among phylogenetically diverse groups of microorganisms. Our findings suggest that microbial communities are capable of repairing D-amino acids within proteins using energy obtained from the degradation of a mixture of labile compounds in microbial necromass and more recalcitrant organic matter.

Bioenergetic Controls on Microbial Ecophysiology in Marine Sediments

Marine sediments constitute one of the most energy-limited habitats on Earth, in which microorganisms persist over extraordinarily long timescales with very slow metabolisms. This habitat provides an ideal environment in which to study the energetic limits of life. However, the bioenergetic factors that can determine whether microorganisms will grow, lie dormant, or die, as well as the selective environmental pressures that determine energetic trade-offs between growth and maintenance activities, are not well understood. Numerical models will be pivotal in addressing these knowledge gaps. However, models rarely account for the variable physiological states of microorganisms and their demand for energy. Here, we review established modeling constructs for microbial growth rate, yield, maintenance, and physiological state, and then provide a new model that incorporates all of these factors. We discuss this new model in context with its future application to the marine subsurface. Understanding the factors that regulate cell death, physiological state changes, and the provenance of maintenance energy (i.e., endogenous versus exogenous metabolism), is crucial to the design of this model. Further, measurements of growth rate, growth yield, and basal metabolic activity will enable bioenergetic parameters to be better constrained. Last, biomass and biogeochemical rate measurements will enable model simulations to be validated. The insight provided from the development and application of new microbial modeling tools for marine sediments will undoubtedly advance the understanding of the minimum power required to support life, and the ecophysiological strategies that organisms utilize to cope under extreme energy limitation for extended periods of time.

Thrifty Females, Frisky Males: Winter Energetics of Hibernating Bats from a Cold Climate.

Mammalian hibernation consists of energy-saving torpor bouts (periods of controlled reduction in body temperature [Tb]) interspersed with brief arousals to normothermic Tb. Frequency and duration of torpor bouts and arousals can affect winter survival and are thought to be influenced by an optimization balancing the energetic benefits of prolonged torpor against the physiological and ecological costs (e.g., accumulation of metabolic wastes). Female little brown bats (Myotis lucifugus) spend their fat reserves more slowly than males during winter, presumably so they can emerge from hibernation in good condition to initiate pregnancy. We used temperature telemetry over three winters to test a prediction of the optimization hypothesis that female M. lucifugus would use longer torpor bouts and/or shorter arousals than males. Females did conserve energy relative to males by adjusting the magnitude and duration of arousals but not the duration of torpor bouts. Although torpor bout duration did not vary by sex for adults, it did vary by age. Adults initially used longer torpor bouts than young-of-the-year, but this difference declined as cave temperature warmed in spring. Males and females in better condition spent more energy during hibernation, again via increased arousal duration rather than decreased torpor bout duration. Longer arousals by males could increase reproductive fitness if males mate with torpid females throughout winter. Our results highlight demographic differences in winter behavior for small hibernators facing extreme energy limitation in cold climates and illustrate the influence that reproductive costs have on hibernation energetics.

EEWNSN: Energy Efficient Wireless Nano Sensor Network MAC Protocol for Communications in the Terahertz Band

Wireless Nano Sensor Networks (WNSNs) are a dense deployment of nano sensors that communicate through electromagnetic waves over the Terahertz band (0.1–10 THz). The extreme energy, processing power and memory capacity limitation of nano-scale devices and the peculiarities of high operating frequency introduce the requirement to design novel communication paradigm and light MAC protocols for WNSNs. In this paper, we present a new Energy Efficient Wireless Nano Sensor Network MAC protocol (EEWNSN-MAC) for mobile multi-hop wireless nanonetworks. The proposed protocol takes advantage of the clustering mechanism and TDMA scheduling scheme to alleviate the mobility effects and transmission collisions. We evaluate performance of the EEWNSN-MAC protocol compared with a similar previously proposed nano-MAC protocol called “Smart-MAC”. For this purpose, we utilize a new NS-3 simulator module named nano-sim. This evaluation is done for three critical metrics in WNSN, namely, the total energy that is consumed per sent/received packet on the network, the packet loss ratio (PLR) and scalability. Finally, the simulation results demonstrate that the EEWNSN-MAC protocol improves the network performance in terms of energy consumption and PLR and it is more scalable compared to the “Smart-MAC” algorithm.

Microbial community assembly and evolution in subseafloor sediment

Bacterial and archaeal communities inhabiting the subsurface seabed live under strong energy limitation and have growth rates that are orders of magnitude slower than laboratory-grown cultures. It is not understood how subsurface microbial communities are assembled and whether populations undergo adaptive evolution or accumulate mutations as a result of impaired DNA repair under such energy-limited conditions. Here we use amplicon sequencing to explore changes of microbial communities during burial and isolation from the surface to the >5,000-y-old subsurface of marine sediment and identify a small core set of mostly uncultured bacteria and archaea that is present throughout the sediment column. These persisting populations constitute a small fraction of the entire community at the surface but become predominant in the subsurface. We followed patterns of genome diversity with depth in four dominant lineages of the persisting populations by mapping metagenomic sequence reads onto single-cell genomes. Nucleotide sequence diversity was uniformly low and did not change with age and depth of the sediment. Likewise, there was no detectable change in mutation rates and efficacy of selection. Our results indicate that subsurface microbial communities predominantly assemble by selective survival of taxa able to persist under extreme energy limitation.

Size and Carbon Content of Sub-seafloor Microbial Cells at Landsort Deep, Baltic Sea.

The discovery of a microbial ecosystem in ocean sediments has evoked interest in life under extreme energy limitation and its role in global element cycling. However, fundamental parameters such as the size and the amount of biomass of sub-seafloor microbial cells are poorly constrained. Here we determined the volume and the carbon content of microbial cells from a marine sediment drill core retrieved by the Integrated Ocean Drilling Program (IODP), Expedition 347, at Landsort Deep, Baltic Sea. To determine their shape and volume, cells were separated from the sediment matrix by multi-layer density centrifugation and visualized via epifluorescence microscopy (FM) and scanning electron microscopy (SEM). Total cell-carbon was calculated from amino acid-carbon, which was analyzed by high-performance liquid chromatography (HPLC) after cells had been purified by fluorescence-activated cell sorting (FACS). The majority of microbial cells in the sediment have coccoid or slightly elongated morphology. From the sediment surface to the deepest investigated sample (~60 m below the seafloor), the cell volume of both coccoid and elongated cells decreased by an order of magnitude from ~0.05 to 0.005 μm3. The cell-specific carbon content was 19–31 fg C cell−1, which is at the lower end of previous estimates that were used for global estimates of microbial biomass. The cell-specific carbon density increased with sediment depth from about 200 to 1000 fg C μm−3, suggesting that cells decrease their water content and grow small cell sizes as adaptation to the long-term subsistence at very low energy availability in the deep biosphere. We present for the first time depth-related data on the cell volume and carbon content of sedimentary microbial cells buried down to 60 m below the seafloor. Our data enable estimates of volume- and biomass-specific cellular rates of energy metabolism in the deep biosphere and will improve global estimates of microbial biomass.

Life under extreme energy limitation: a synthesis of laboratory- and field-based investigations.

The ability of microorganisms to withstand long periods with extremely low energy input has gained increasing scientific attention in recent years. Starvation experiments in the laboratory have shown that a phylogenetically wide range of microorganisms evolve fitness-enhancing genetic traits within weeks of incubation under low-energy stress. Studies on natural environments that are cut off from new energy supplies over geologic time scales, such as deeply buried sediments, suggest that similar adaptations might mediate survival under energy limitation in the environment. Yet, the extent to which laboratory-based evidence of starvation survival in pure or mixed cultures can be extrapolated to sustained microbial ecosystems in nature remains unclear. In this review, we discuss past investigations on microbial energy requirements and adaptations to energy limitation, identify gaps in our current knowledge, and outline possible future foci of research on life under extreme energy limitation.

A low phosphorylation potential in the acetogen Acetobacterium woodii reflects its lifestyle at the thermodynamic edge of life.

The anaerobic, acetogenic bacterium Acetobacterium woodii grows on hydrogen and carbon dioxide and uses the Wood-Ljungdahl pathway to fix carbon but also to synthesize ATP. The free energy change of acetogenesis from H2+CO2 allows for synthesis of only a fraction of an ATP under environmental conditions, and A. woodii is clearly a paradigm for microbial life under extreme energy limitation. However, it was unknown how much energy is required to make ATP under these conditions. In the present study, we determined the phosphorylation potential in cells metabolizing three different acetogenic substrates. It accounts to 37.9±1.3kJ/mol ATP during acetogenesis from fructose, 32.1±0.3kJ/mol ATP during acetogenesis from H2+CO2 and 30.2±0.9kJ/mol ATP during acetogenesis from CO, the lowest phosphorylation potential ever described. The physiological consequences in terms of energy conservation under extreme energy limitation are discussed.

Warming up and shipping out: arousal and emergence timing in hibernating little brown bats (Myotis lucifugus).

Phenology refers to the timing of events in the annual cycle of organisms. For temperate-zone mammals, hibernation is one such event, but little is known about its phenology. Hibernation consists of energy-saving torpor bouts interspersed with energetically expensive arousals to normothermic Tb, and hibernators should benefit from mechanisms which reduce arousal costs and help them time arousals to coincide with foraging opportunities. In a previous study, we showed that, in contrast to hibernating bats from warmer climates, little brown bats (Myotis lucifugus) from central Canada abandon a circadian pattern to arousal in the middle of winter when there is no chance of feeding. Here, we used temperature telemetry to test whether they would re-synchronize arousals with normal foraging time (i.e. sunset) during late winter as the chance of foraging or emergence opportunities improves, and whether they would synchronize arousals with conspecifics, possibly to exploit social thermoregulation. We also used passive transponders to test whether energy reserves and/or sex differences in reproductive timing influence phenology and the sensitivity of emergence timing to environmental cues. In contrast to patterns in mid-winter, after 7 April 2013, bats synchronized arousals with sunset and with conspecifics. Females emerged earlier than males, and females in the best condition emerged first while body condition had no influence on male emergence timing. Both male and female bats appeared to time emergence with falling barometric pressure, a cue that predicts favourable foraging conditions for bats but which, unlike outside temperature, would have been readily detectable by bats inside the hibernaculum. Our results highlight hibernation traits associated with extreme winter energy limitation for insect-eating bats in cold climates and illustrate the influence of reproductive timing and environmental conditions on hibernation energetics and phenology.

Adaptations of anaerobic archaea to life under extreme energy limitation

Some anaerobic archaea live on substrates that do not allow the synthesis of 1 mol of ATP per mol of substrate. Energy conservation in these cases is only possible by a chemiosmotic mechanism that involves the generation of an electrochemical ion gradient across the cytoplasmatic membrane that then drives ATP synthesis via an A1AO ATP synthase. The minimal amount of energy required is thus depending on the magnitude of the electrochemical ion gradient, the phosphorylation potential, and the ion/ATP ratio of the ATP synthase. Methanogens, Thermococcus, Pyrococcus, and Ignicoccus have evolved different ways to energize their membranes, such as methyltransferases, H+, or NAD+ reducing electron transport systems fueled by reduced ferredoxin or H2 -dependent sulfur reduction that all operate at the thermodynamic limit of life. The structure and function of the enzymes involved are discussed. Despite the differences in membrane energization, they have in common an A1AO ATP synthase that shows an extraordinary divergence in rotor composition and structural adaptations to life under these conditions. In sum, adaptation of anaerobic archaea to energy-limited substrates involves chemiosmotic energy coupling, often with Na+ as coupling ion and a structurally and functionally highly adapted ATP synthase.

Surviving the Cold, Dry Period in Africa: Behavioral Adjustments as an Alternative to Heterothermy in the African Lesser Bushbaby (Galago moholi)

Behavioral and physiological adaptations are common and successful strategies used by small endothermic species to adjust to unfavorable seasons. Physiological adaptations, such as heterothermy, e.g., torpor, are usually thought to be more effective energy-saving strategies than behavioral adjustments. The African lesser bushbaby, Galago moholi, is physiologically capable of torpor but expresses heterothermy only under conditions of extreme energy limitation, suggesting that it has evolved alternative strategies to compensate energetic bottlenecks. We hypothesized that Galagomoholi survives the unfavorable winter period, without —or only rarely— employing torpid phases to save energy, by using behavioral thermoregulation. We compared the ecology and behavior of Galago moholi in summer and winter by telemetric tracking and examined food availability by determination of arthropod and gum availability. We found a significant increase in huddling behavior and a significant increase in the use of enclosed and insulated sleeping sites during winter, as well as a reduction in nightly activity. Galago moholi hunted for insects significantly less in winter than in summer, and increased gum intake in winter, when gum showed an increase in energy content. The availability of high-quality food, albeit in low quantities, presumably enables Galago moholi to stay normothermic throughout the cold, dry period and to focus on reproduction activities. We propose that Galago moholi favors ecological and behavioral adjustments over torpor because these are sufficient to meet energy requirements of this species, and their advantages (flexibility, unrestricted activity, and reproduction) outweigh the energetic benefits of heterothermy.

Electron transport during aceticlastic methanogenesis by Methanosarcina acetivorans involves a sodium‐translocating Rnf complex

The anaerobic methanogenic archaeon Methanosarcinaacetivorans lives under extreme energy limitation. Methanogenesis from acetate as carried out by M.acetivorans involves an anaerobic electron transport chain with ferredoxin as electron donor and heterodisulfide as electron acceptor, and so far only the heterodisulfide reductase has been shown to translocate H(+) . Here, we describe a second Na(+) -translocating coupling site in this electron transport chain. Inside-out membrane vesicles of M.acetivorans catalyzed Na(+) transport coupled to an electron transport catalyzed by the ferredoxin:heterodisulfide oxidoreductase activity. Ionophore studies revealed that Na(+) transport was primary and electrogenic. A ∆rnf mutant was unable to grow on acetate and the ferredoxin:heterodisulfide oxidoreductase-coupled Na(+) transport was abolished. These data are consistent with the hypothesis that the Rnf complex of M.acetivorans is an Na(+) -translocating coupling site and the entry point of electrons derived from reduced ferredoxin into the electron transport chain leading to the heterodisulfide.

Feast and famine — microbial life in the deep-sea bed

The seabed is a diverse environment that ranges from the desert-like deep seafloor to the rich oases that are present at seeps, vents, and food falls such as whales, wood or kelp. As well as the sedimentation of organic material from above, geological processes transport chemical energy--hydrogen, methane, hydrogen sulphide and iron--to the seafloor from the subsurface below, which provides a significant proportion of the deep-sea energy. At the sites on the seafloor where chemical energy is delivered, rich and diverse microbial communities thrive. However, most subsurface microorganisms live in conditions of extreme energy limitation, with mean generation times of up to thousands of years. Even in the most remote subsurface habitats, temperature rather than energy seems to set the ultimate limit for life, and in the deep biosphere, where energy is most depleted, life might even be based on the cleavage of water by natural radioisotopes. Here, we review microbial biodiversity and function in these intriguing environments.