Bolbfish The blobfish (Psychrolutes marcidus) is a fish that inhabits the deep waters off the coasts of Australia and Tasmania. Due to the inaccessibility of its habitat, it is rarely seen by humans. Blobfish are found at depths greater than 5000 m, which would likely make gas bladders inefficient. To remain buoyant, the flesh of the blobfish is primarily a gelatinous mass with a density slightly less than water; this allows the fish to float above the sea floor without expending energy on swimming. The relative lack of muscle is not a disadvantage as it primarily swallows edible matter that floats by in front it

Key Questions What are the different biomes that are important to the deep carbon cycle? Terrestrial marine What is the magnitudes, rates and kinds of microbial activity in the different biomes? Temporal/spatial scales What are the sources and sinks of organic carbon in deep environments (biotic, abiotic, and modified)? What limits deep life? Coupled temp-pressure- energy- porosity/perm.

Terrestrial Biomes Many are hydrogen driven systems

Terrestrial subsurface SLiME - subsurface lithoautotrophic microbial ecosystems Deep cratons Columbia River basalts Nealson et al. 2005

Marine biomes Complex sources and sinks for carbon Pelagic environments Light-driven and dark CO2 fixation Carbon flux to benthos, crust etc Deep sediments Hydrothermal vents and subseafloor crust New eruptions and linkages Rock hosted including the deep subseafloor Subduction zones

Subseafloor fluid flow regimes + settings fluid transport = life JOIDES Hydrogeology PPG Report (2001)

Taking the Pulse of a Plate: Hydrogeological-Biological Observatories There are over 15,000 seamounts -hydrothermal “breathing” holes? The oceanic crust is the largest fractured aquifer on Earth 85% of the Earths magmatic budget is focused at mid-ocean ridges The margins host ~10,000 gigatons of hydrate The subseafloor biosphere may rival that on the continents?

Sources and sinks of carbon

Size spectrum of organic matter and other “things” in the ocean From Verdugo 2004

Colloids in the marine environment: the most abundant form of carbon Colloids range in size from extremely small (5-200 nm) to large (0.4-1µm). Small colloids are more abundant and can reach 109/ml whereas the larger colloids are less abundant (~107/ml) Most of the colloids are refractory carbohydrates There are multiple sources for colloids Nothing known about the the possible degradation of colloids and the role bacteria play in production and consumption

Depth distribution of small (5-200 nm) colloid particles-concentrations (X 109 ml-1) from Wells and Goldberg, 1994

From Wells and Goldberg, 1994

Incidence, diversity and physiology of “deep” microbial communities Incidence and diversity Metabolism of CO2 fixing microbes Physiology of isolated microorganisms

Number and metabolic diversity of microorganisms in vent and other deep-sea environments Samples Number of microorganisms Metabolic and/or phylogenetic groups Sulfide structures >108 per gram sulfide on outer layers; 105 per gram in interior Outer layers have both bacteria and archaea and include metal oxidizers and methanogens; inner layers contain archaea of unknown physiologies Diffuse-flow fluids (2°C to ~80°C) 105->109 ml-1; high numbers from Galapagos particles Extremely high diversity of bacteria and archaea (all thermal groups) Smoker fluids (<200°C to ~400°C) Not detected to 107 ml-1; high numbers correlate with phase separation Hyperthermophilic bacteria and archaea from culture and molecular analyses Hydrothermal vent plume water (2°C in horizontal plume ~105 to >106 ml-1 H2, CH4 and Mn2+ oxidizing bacteria detected by activity measurements Deep SW surrounding vents (2°C) 103 to <105 ml-1 Limited diversity of bacteria and archaea detected and enumerated by molecular methods

Number of microorganisms Metabolic and/or phylogenetic groups Number and metabolic diversity of microorganisms in deep-sea environments - continued Samples Number of microorganisms Metabolic and/or phylogenetic groups Subseafloor crust Numbers unknown on axis; ~105 ml-1 in old crust (>4 Ma) Different thermal groups of bacteria and archaea detected from new eruptions; unique archaea isolated from subsurface fluids Microbial mats >108 bacteria per gram High numbers of S-oxidizing bacteria including Beggiatoa spp and uncultured -Proteobacteria Sediments >108 bacteria per gram in the surface decreasing numbers with depth Same as for microbial mats in surface layer with sulfate-reducing bacteria and methanogens dominating the deeper layers

The Primary Producers

Questions and Issues - I: Primary Production What is the phylogenetic and physiological diversity of the primary producers in deep-sea environments (deep sediments, crust, diffuse flow vents, sulfides, animal symbionts, plumes, microbial mats, etc)? What is metabolic versatility of the primary producers? (CO2 fixation) How significant is the abiotic synthesis of organic compounds (C1 - Cn) to primary production? (coupling the oxidation of organic compounds with the reduction of FeIII and S°) How do the primary producers effect biogeochemical cycles (Metal, S, P and N)? What is the primary production rates in situ in different vent environments? What is the diversity of N2 fixing microorganisms and how important is nitrogen fixation to primary production? What are the sources and sinks for biologically utilizable phosphate?

Ax99-59 isolated from Axial Volcano Strict anaerobe Thermophilic CO2 is carbon source H2 as energy source Reduces sulfur species 32 min doubling time under optimal conditions G+C ratio if 40% New genus in the Aquifacales *Also -Proteobacteria are important primary producers Scanning electron micrograph of Ax99-59. Under most culturing conditions this organism produce copious amount of exo- polysaccharide, which may be involved in Biofilm formation. Scale bar is 1 µm Huber, unpublished

PNAS 102:9306-9310, 2005 A Green-sulfur photosynthetic bacteria was isolated from a submarine hydrothermal vent smoker where the only source of light is geothermal radiation that includes wavelengths absorbed by photosynthetic pigments. This organisms is an obligate anaerobe and reduces CO2 coupled with oxidation of sulfur compounds Photosynthetic bacteria Chlorosomes Morphology and ultrastructure of GSB1 cells. Bar, 300 nm 2HCO3- + H2S  2CH2O + SO42-

Experiments Design experiments to investigate the effect of spatial gradients on microbial activity Laying the groundwork for doing focused experimental studies (with potential industrial/societal/environmental impacts) Better descriptions of physiology of microbes Experiments to better understand OM processing at high temperatures and pressures versus transformations to acetate, methane, etc. Relate microbial physiology to the carbon budget at organism to community scales.

Fieldwork Some environments are readily accessible and some require longer term planning and how best to sample them) 85% of magmatic budget focused at ridge, but only 2 actual observations- need more data! There are heterotrophs in deep subsurface environments (deep OM processing) Organic sources are potentially metabolites of the autotrophs Need to delineate sources of metabolites How many spores are we missing?/or cyst-like states (survival)

Conclusions and Implications Astrobiology (ice habitats and impact sites) Origin of life and paleo issues Metabolism vs. time Physiology a. Metabolism b. Survival strategies/stress responses c. Consortial strategies d. Genome evolutions (HGT) Need to better define and delineate deep life and deep habitats Possible applications Sequestration, biofuels, etc

From Martin, Baross, Kelley and Russell, Nature Microbiology Rev From Martin, Baross, Kelley and Russell, Nature Microbiology Rev. submitted