deep biosphere deep biosphere the deep biosphere is the part of the biosphere that resides below the first few meters of the surface. it extends down at least 5 kilometers below the continental surface and 10.5 kilometers below the sea surface, at temperatures that may reach beyond 120 °c, which is comparable to the maximum temperature where a metabolically active organism has been found. it includes all three domains of life and the genetic diversity rivals that on the surface. the first indications of deep life came from studies of oil fields in the 1920s, but it was not certain that the organisms were indigenous until methods were developed in the 1980s to prevent contamination from the surface. samples are now collected in deep mines and scientific drilling programs in the ocean and on land. deep observatories have been established for more extended studies. near the surface, living organisms consume organic matter and breathe oxygen. lower down, these are not available, so they make use of "edibles" (electron donors) such as hydrogen (released from rocks by various chemical processes), methane (ch4), reduced sulfur compounds, and ammonium (nh4). they "breathe" electron acceptors such as nitrates and nitrites, manganese and iron oxides, oxidized sulfur compounds and carbon dioxide (co2). there is very little energy at greater depths, so metabolisms are up to a million times slower than at the surface. cells may live for thousands of years before dividing and there is no known limit to their age. the subsurface accounts for about 90% of the biomass in two domains of life, archaea and bacteria, and 15% of the total for the biosphere. eukarya are also found, including some multicellular life fungi, and animals (nematodes, flatworms, rotifers, annelids, and arthropods). viruses are also present and infect the microbes. definition the deep biosphere is an ecosystem of organisms and their living space in the deep subsurface. for the seafloor, an operational definition of deep subsurface is the region that is not bioturbated by animals; this is generally about a meter or more below the surface. on continents, it is below a few meters, not including soils. the organisms in this zone are sometimes referred to as intraterrestrials. history at the university of chicago in the 1920s, geologist edson bastin enlisted the help of microbiologist frank greer in an effort to explain why water extracted from oil fields contained hydrogen sulfide and bicarbonates. these chemicals are normally created by bacteria, but the water came from a depth where the heat and pressure were considered too great to support life. they were able to culture anaerobic sulfate-reducing bacteria from the water, demonstrating that the chemicals had a bacterial origin. also in the 1920s, charles lipman, a microbiologist at the university of california, berkeley, noticed that bacteria that had been sealed in bottles for 40 years could be reanimated – a phenomenon now known as anhydrobiosis. he wondered whether the same was true of bacteria in coal seams. he sterilized samples of coal, wetted them, crushed them and then succeeded in culturing bacteria from the coal dust. one sterilization procedure, baking the coal at 160 degrees celsius for up to 50 hours, actually encouraged their growth. he published the results in 1931. the first studies of subsurface life were conducted by claude e. zobell, the "father of marine microbiology", in the late 1930s to the 1950s. although the coring depth was limited, microbes were found wherever the sediments were sampled. with increasing depth, aerobes gave way to anaerobes. photograph of the sunken alvin taken in 1969 most biologists dismissed the subsurface microbes as contamination, especially after the submersible alvin sank in 1968 and the scientists escaped, leaving their lunches behind. when alvin was recovered, the lunches showed no sign of microbial decay. this reinforced a view of the deep sea (and by extension the subsurface) as a lifeless desert. the study of the deep biosphere was dormant for decades, except for some soviet microbiologists who began to refer to themselves as geomicrobiologists. interest in subsurface life was renewed when the united states department of energy was looking for a safe way of burying nuclear waste, and frank j. wobber realized that microbes below the surface could either help by degrading the buried waste or hinder by breaching the sealed containers. he formed the subsurface science program to study deep life. to address the problem of contamination, special equipment was designed to minimize contact between a core sample and the drilling fluid that lubricates the drill bit. in addition, tracers were added to the fluid to indicate whether it penetrated the core. in 1987, several boreholes were drilled near the savannah river site, and microorganisms were found to be plentiful and diverse at least 500 metres below the surface. from 1983 until now, microbiologists analyze cell abundances in drill cores from the ocean drilling program (now the international ocean discovery program). a group led by john parkes of the university of bristol reported concentrations of 104 to 108 cells per gram of sediment down to depths of 500 metres (agricultural soils contain about 109 cells per gram). this was initially met with skepticism and it took them four years to publish their results. in 1998, william whitman and colleagues published a summary of twelve years of data in the proceedings of the national academy of sciences. they estimated that up to 95% of all prokaryotes (archaea and bacteria) live in the deep subsurface, with 55% in the marine subsurface and 39% in the terrestrial subsurface. in 2002, ocean drilling program leg 201 was the first to be motivated by a search for deep life. most of the previous exploration was on continental margins, so the goal was to drill in the open ocean for comparison. in 2016, international ocean discovery program leg 370 drilled into the marine sediment of the nankai accretionary prism and observed 102 vegetative cells at 118 °c. understanding of microbial life at depth was extended beyond the realm of oceanography to earth sciences (and even astrobiology) in 1992 when thomas gold published a paper in the proceedings of the national academy of sciences that was titled "the deep, hot biosphere", followed seven years later by his book, the deep hot biosphere. a 1993 article by journalist william broad, published in the new york times and titled "strange new microbes hint at a vast subterranean world," carried gold's thesis to public attention. the article began, "new forms of microbial life are being discovered in such abundance deep inside the earth that some scientists are beginning to suspect that the planet has a hidden biosphere extending miles down whose total mass may rival or exceed that of all surface life. if a deep biosphere does exist, scientists say, its discovery will rewrite textbooks while shedding new light on the mystery of life's origins. even skeptics say the thesis is intriguing enough to warrant new studies of the subterranean realm." the 1993 article also features how gold's thesis expands possibilities for astrobiology research: "dr. thomas gold, an astrophysicist at cornell university known for bold theorizing, has speculated that subterranean life may dot the cosmos, secluded beneath the surfaces of planets and moons and energized by geological processes, with no need for the warming radiation of nearby stars. he wrote in the proceedings of the national academy of sciences last year that the solar system might harbor at least 10 deep biospheres. 'such life may be widely disseminated in the universe,' he said, 'since planetary type bodies with similar subsurface conditions may be common as solitary objects in space, as well as in other solar-type systems.'" freeman dyson wrote the foreword to gold's 1999 book, where he concluded, "gold's theories are always original, always important, usually controversial — and usually right. it is my belief, based on fifty years of observation of gold as a friend and colleague, that the deep hot biosphere is all of the above: original, important, controversial — and right." dyson also delivered a eulogy at gold's memorial service in 2004, a segment of which pertaining to the deep hot biosphere theory is posted on youtube. following gold's death, scientific discoveries amplified and also shifted understanding of the deep biosphere. a term gold coined in his 1999 book, however, carries forward and is a reminder of the worldview shift he advocated. the term is "surface chauvinism". gold wrote, "in retrospect, it is not hard to understand why the scientific community has typically sought only surface life in the heavens. scientists have been hindered by a sort of 'surface chauvinism.'". scientific methods the present understanding of subsurface biology was made possible by numerous advances in technology for sample collection, field analysis, molecular science, cultivation, imaging and computation. sample collection schematic of an expedition aboard the japanese drilling ship d/v chikyū. researcher sampling fluid from a deep mine. the ocean floor is sampled by drilling boreholes and collecting cores. the methods must be adapted to different types of rock, and the cost of drilling limits the number of holes that can be drilled. microbiologists have made use of scientific drilling programs: the ocean drilling program (odp), which used the joides resolution drilling platform, and the integrated ocean drilling program (iodp), which used the japanese ship chikyū. deep underground mines, for example south african gold mines and the pyhäsalmi copper and zinc mine in finland, have provided opportunities to sample the deep biosphere. the deep subsurface has also been sampled at chosen or proposed nuclear waste repository sites (e.g. yucca mountain and the waste isolation pilot plant in the united states, äspö and surrounding areas in sweden, onkalo and surrounding areas in finland, mont terri in switzerland). scientific drilling of continental deep subsurface has been promoted by the international continental scientific drilling program (icdp). to allow continuous underground sampling, various kinds of observatories have been developed. on the ocean floor, the circulation obviation retrofit kit (cork) seals a borehole to cut off the influx of seawater. an advanced version of cork is able to seal off multiple sections of a drill hole using packers, rubber tubes that inflate to seal the space between the drill string and the wall of the borehole. in sediments, the simple cabled instrument for measuring parameters in-situ (scimpi) is designed to remain and take measurements after a borehole has collapsed. packers are also used in the continental subsurface, along with devices such as the flow-through in situ reactor (ftisr). various methods are used to extract fluids from these sites, including passive gas samplers, u-tube systems and osmotic gas samplers. in narrow (less than 50 mm) holes, polyamide tubes with a back-pressure valve can be lowered to sample an entire column of fluid. field analysis and manipulation some methods analyze microbes in situ rather than extract them. in biogeophysics, the effects of microbes on properties of geological materials are remotely probed using electrical signals. microbes can be tagged using a stable isotope such as carbon-13 and then re-injected in the ground to see where they go. a "push-pull" method involves injection of a fluid into an aquifer and extraction of a mixture of injected fluid with the ground water; the latter can then be analyzed to determine what chemical reactions occurred. molecular methods and cultivation methods from modern molecular biology allow the extraction of nucleic acids, lipids and proteins from cells, dna sequencing, and the physical and chemical analysis of molecules using mass spectrometry and flow cytometry. a lot can be learned about the microbial communities using these methods even when the individuals cannot be cultivated. for example, at the richmond mine in california, scientists used shotgun sequencing to identify four new species of bacteria, three new species of archaea (known as the archaeal richmond mine acidophilic nanoorganisms), and 572 proteins unique to the bacteria. geochemical methods deep microorganisms change the chemistry of their surroundings. they consume nutrients and product wastes from metabolism. therefore we can estimate the activities of the deep microorganisms by measuring the chemical compositions in the subseafloor samples. complementary techniques include measuring the isotope compositions of the chemicals or the related minerals. conditions for life for life to have metabolic activity, it must be able to take advantage of a thermodynamic disequilibrium in the environment. this can occur when rocks from the mantle that are rich