Douglas E. LaRowe

Microbial activity in the marine deep biosphere: progress and prospects

The vast marine deep biosphere consists of microbial habitats within sediment, pore waters, upper basaltic crust and the fluids that circulate throughout it. A wide range of temperature, pressure, pH, and electron donor and acceptor conditions exists—all of which can combine to affect carbon and nutrient cycling and result in gradients on spatial scales ranging from millimeters to kilometers. Diverse and mostly uncharacterized microorganisms live in these habitats, and potentially play a role in mediating global scale biogeochemical processes. Quantifying the rates at which microbial activity in the subsurface occurs is a challenging endeavor, yet developing an understanding of these rates is essential to determine the impact of subsurface life on Earths global biogeochemical cycles, and for understanding how microorganisms in these “extreme” environments survive (or even thrive). Here, we synthesize recent advances and discoveries pertaining to microbial activity in the marine deep subsurface, and we highlight topics about which there is still little understanding and suggest potential paths forward to address them. This publication is the result of a workshop held in August 2012 by the NSF-funded Center for Dark Energy Biosphere Investigations (C-DEBI) “theme team” on microbial activity (www.darkenergybiosphere.org).

The energetics of organic synthesis inside and outside the cell

Thermodynamic modelling of organic synthesis has largely been focused on deep-sea hydrothermal systems. When seawater mixes with hydrothermal fluids, redox gradients are established that serve as potential energy sources for the formation of organic compounds and biomolecules from inorganic starting materials. This energetic drive, which varies substantially depending on the type of host rock, is present and available both for abiotic (outside the cell) and biotic (inside the cell) processes. Here, we review and interpret a library of theoretical studies that target organic synthesis energetics. The biogeochemical scenarios evaluated include those in present-day hydrothermal systems and in putative early Earth environments. It is consistently and repeatedly shown in these studies that the formation of relatively simple organic compounds and biomolecules can be energy-yielding (exergonic) at conditions that occur in hydrothermal systems. Expanding on our ability to calculate biomass synthesis energetics, we also present here a new approach for estimating the energetics of polymerization reactions, specifically those associated with polypeptide formation from the requisite amino acids.

Thermodynamic Potential for the Abiotic Synthesis of Adenine, Cytosine, Guanine, Thymine, Uracil, Ribose, and Deoxyribose in Hydrothermal Systems

The thermodynamic potential for the abiotic synthesis of the five common nucleobases (adenine, cytosine, guanine, thymine, and uracil) and two monosaccharides (ribose and deoxyribose) from formaldehyde and hydrogen cyanide has been quantified under temperature, pressure, and bulk composition conditions that are representative of hydrothermal systems. The activities of the precursor molecules (formaldehyde and hydrogen cyanide) required to evaluate the thermodynamics of biomolecule synthesis were computed using the concentrations of aqueous N2, CO, CO2 and H2 reported in the modern Rainbow hydrothermal system. The concentrations of precursor molecules that can be synthesized are strongly dependent on temperature with larger concentrations prevailing at lower temperatures. Similarly, the thermodynamic drive to synthesize nucleobases, ribose and deoxyribose varies considerably as a function of temperature: all of the biomolecules considered in this study are thermodynamically favored to be synthesized throughout the temperature range from 0°C to between 150°C and 250°C, depending on the biomolecule. Furthermore, activity diagrams have been generated to illustrate that activities in the range of 10−2– 10−6 for nucleobases, ribose and deoxyribose can be in equilibrium with a range of precursor molecule activities at 150°C and 500 bars. The results presented here support the notion that hydrothermal systems could have played a fundamental role in the origin of life, and can be used to plan and constrain experimental investigation of the abiotic synthesis of nucleic-acid related biomolecules.

Biosphere frontiers of subsurface life in the sedimented hydrothermal system of Guaymas Basin.

Temperature is one of the key constraints on the spatial extent, physiological and phylogenetic diversity, and biogeochemical function of subsurface life. A model system to explore these interrelationships should offer a suitable range of geochemical regimes, carbon substrates and temperature gradients under which microbial life can generate energy and sustain itself. In this theory and hypothesis article, we make the case for the hydrothermally heated sediments of Guaymas Basin in the Gulf of California as a suitable model system where extensive temperature and geochemical gradients create distinct niches for active microbial populations in the hydrothermally influenced sedimentary subsurface that in turn intercept and process hydrothermally generated carbon sources. We synthesize the evidence for high-temperature microbial methane cycling and sulfate reduction at Guaymas Basin – with an eye on sulfate-dependent oxidation of abundant alkanes – and demonstrate the energetic feasibility of these latter types of deep subsurface life in previously drilled Guaymas Basin locations of Deep-Sea Drilling Project 64.

A thermodynamic analysis of the anaerobic oxidation of methane in marine sediments

Anaerobic oxidation of methane (AOM) in anoxic marine sediments is a significant process in the global methane cycle, yet little is known about the role of bulk composition, temperature and pressure on the overall energetics of this process. To better understand the biogeochemistry of AOM, we have calculated and compared the energetics of a number of candidate reactions that microorganisms catalyse during the anaerobic oxidation of methane in (i) a coastal lagoon (Cape Lookout Bight, USA), (ii) the deep Black Sea, and (iii) a deep-sea hydrothermal system (Guaymas basin, Gulf of California). Depending on the metabolic pathway and the environment considered, the amount of energy available to the microorganisms varies from 0 to 184 kJ mol(-1). At each site, the reactions in which methane is either oxidized to HCO3(-), acetate or formate are generally only favoured under a narrow range of pressure, temperature and solution composition--particularly under low (10(-10 )m) hydrogen concentrations. In contrast, the reactions involving sulfate reduction with H2, formate and acetate as electron donors are nearly always thermodynamically favoured. Furthermore, the energetics of ATP synthesis was quantified per mole of methane oxidized. Depending on depth, between 0.4 and 0.6 mol of ATP (mol CH4(-1) was produced in the Black Sea sediments. The largest potential productivity of 0.7 mol of ATP (mol CH4(-1) was calculated for Guaymas Basin, while the lowest values were predicted at Cape Lookout Bight. The approach used in this study leads to a better understanding of the environmental controls on the energetics of AOM.

Chemolithotrophy in the continental deep subsurface: Sanford Underground Research Facility (SURF), USA

The deep subsurface is an enormous repository of microbial life. However, the metabolic capabilities of these microorganisms and the degree to which they are dependent on surface processes are largely unknown. Due to the logistical difficulty of sampling and inherent heterogeneity, the microbial populations of the terrestrial subsurface are poorly characterized. In an effort to better understand the biogeochemistry of deep terrestrial habitats, we evaluate the energetic yield of chemolithotrophic metabolisms and microbial diversity in the Sanford Underground Research Facility (SURF) in the former Homestake Gold Mine, SD, USA. Geochemical data, energetic modeling, and DNA sequencing were combined with principle component analysis to describe this deep (down to 8100 ft below surface), terrestrial environment. SURF provides access into an iron-rich Paleoproterozoic metasedimentary deposit that contains deeply circulating groundwater. Geochemical analyses of subsurface fluids reveal enormous geochemical diversity ranging widely in salinity, oxidation state (ORP 330 to −328 mV), and concentrations of redox sensitive species (e.g., Fe2+ from near 0 to 6.2 mg/L and Σ S2- from 7 to 2778μg/L). As a direct result of this compositional buffet, Gibbs energy calculations reveal an abundance of energy for microorganisms from the oxidation of sulfur, iron, nitrogen, methane, and manganese. Pyrotag DNA sequencing reveals diverse communities of chemolithoautotrophs, thermophiles, aerobic and anaerobic heterotrophs, and numerous uncultivated clades. Extrapolated across the mine footprint, these data suggest a complex spatial mosaic of subsurface primary productivity that is in good agreement with predicted energy yields. Notably, we report Gibbs energy normalized both per mole of reaction and per kg fluid (energy density) and find the later to be more consistent with observed physiologies and environmental conditions. Further application of this approach will significantly expand our understanding of the deep terrestrial biosphere.

Catabolic rates, population sizes and doubling/replacement times of microorganisms in natural settings

Directly assessing the impact of subsurface microbial activity on global element cycles is complicated by the inaccessibility of most deep biospheres and the difficulty of growing representative cultivars in the laboratory. In order to constrain the rates of biogeochemical processes in such settings, a quantitative relationship between rates of microbial catalysis, energy supply and demand and population size has been developed that complements the limited biogeochemical data describing subsurface environments. Within this formulation, rates of biomass change are determined as a function of the proportion of catabolic power that is converted into anabolism—either new microorganisms or the replacement of existing cell components—and the amount of energy that is required to synthesize biomass. Catabolic power is related to biomass through an energy-based yield coefficient that takes into account the constraints that different environments impose on biomolecule synthesis; this method is compared to other approaches for determining yield coefficients. Furthermore, so-called microbial maintenance energies that have been reported in the literature, which span many orders of magnitude, are reviewed. The equations developed in this study are used to demonstrate the interrelatedness of catabolic reaction rates, Gibbs energy of reaction, maintenance energy, biomass yield coefficients, microbial population sizes and doubling/replacement times. The number of microorganisms that can be supported by particular combinations of energy supply and demand is illustrated as a function of the catabolic rates in marine environments. Replacement/doubling times for various population sizes are shown as well. Finally, cell count and geochemical data describing two marine sedimentary environments in the South Pacific Gyre and the Peru Margin are used to constrain in situ metabolic and catabolic rates. The formulations developed in this study can be used to better define the limits and extent of life because they are valid for any metabolism under any set of conditions.

Activity and phylogenetic diversity of sulfate-reducing microorganisms in low-temperature subsurface fluids within the upper oceanic crust.

The basaltic ocean crust is the largest aquifer system on Earth, yet the rates of biological activity in this environment are unknown. Low-temperature (<100°C) fluid samples were investigated from two borehole observatories in the Juan de Fuca Ridge (JFR) flank, representing a range of upper oceanic basement thermal and geochemical properties. Microbial sulfate reduction rates (SRR) were measured in laboratory incubations with 35S-sulfate over a range of temperatures and the identity of the corresponding sulfate-reducing microorganisms (SRM) was studied by analyzing the sequence diversity of the functional marker dissimilatory (bi)sulfite reductase (dsrAB) gene. We found that microbial sulfate reduction was limited by the decreasing availability of organic electron donors in higher temperature, more altered fluids. Thermodynamic calculations indicate energetic constraints for metabolism, which together with relatively higher cell-specific SRR reveal increased maintenance requirements, consistent with novel species-level dsrAB phylotypes of thermophilic SRM. Our estimates suggest that microbially-mediated sulfate reduction may account for the removal of organic matter in fluids within the upper oceanic crust and underscore the potential quantitative impact of microbial processes in deep subsurface marine crustal fluids on marine and global biogeochemical carbon cycling.

Thermal and geochemical influences on microbial biogeography in the hydrothermal sediments of Guaymas Basin, Gulf of California

Extreme thermal gradients and compressed metabolic zones limit the depth range of microbial colonization in hydrothermally active sediments at Guaymas Basin. We investigated the physicochemical characteristics of this ecosystem and their influence on microbial community structure. Temperature-related trends of δ(13)C values of methane and dissolved inorganic carbon from 36 sediment cores suggest in situ thermal limits for microbial anaerobic methane oxidation and organic carbon re-mineralization near 80°C and 100°C respectively. Temperature logging probes deposited in hydrothermal sediments for 8 days demonstrate substantial thermal fluctuations of up to 25°C. Putative anaerobic methanotroph (ANME) populations dominate the archaeal community, transitioning from ANME-1 archaea in warm surficial sediments towards ANME-1 Guaymas archaea as temperatures increase downcore. Since ANME archaea performing anaerobic oxidation of methane double on longer time scales (months) compared with relatively rapid in situ temperature fluctuations (hours to days), we conclude that ANME archaea possess a high tolerance for short-term shifts in the thermal regime.

Power limits for microbial life

To better understand the origin, evolution, and extent of life, we seek to determine the minimum flux of energy needed for organisms to remain viable. Despite the difficulties associated with direct measurement of the power limits for life, it is possible to use existing data and models to constrain the minimum flux of energy required to sustain microorganisms. Here, a we apply a bioenergetic model to a well characterized marine sedimentary environment in order to quantify the amount of power organisms use in an ultralow-energy setting. In particular, we show a direct link between power consumption in this environment and the amount of biomass (cells cm-3) found in it. The power supply resulting from the aerobic degradation of particular organic carbon (POC) at IODP Site U1370 in the South Pacific Gyre is between ∼10-12 and 10-16 W cm-3. The rates of POC degradation are calculated using a continuum model while Gibbs energies have been computed using geochemical data describing the sediment as a function of depth. Although laboratory-determined values of maintenance power do a poor job of representing the amount of biomass in U1370 sediments, the number of cells per cm-3 can be well-captured using a maintenance power, 190 zW cell-1, two orders of magnitude lower than the lowest value reported in the literature. In addition, we have combined cell counts and calculated power supplies to determine that, on average, the microorganisms at Site U1370 require 50–3500 zW cell-1, with most values under ∼300 zW cell-1. Furthermore, we carried out an analysis of the absolute minimum power requirement for a single cell to remain viable to be on the order of 1 zW cell-1.