The number of prokaryotes and the total amount of their cellular carbon on earth are estimated to be 4-6 3 10 30 cells and 350-550 Pg of C (1 Pg 5 10 15 g), respectively. Thus, the total amount of prokaryotic carbon is 60-100% of the estimated total carbon in plants, and inclusion of prokaryotic carbon in global models will almost double estimates of the amount of carbon stored in living organisms. In addition, the earth's prokaryotes contain 85-130 Pg of N and 9-14 Pg of P, or about 10-fold more of these nutrients than do plants, and represent the largest pool of these nutrients in living organisms. Most of the earth's prokaryotes occur in the open ocean, in soil, and in oceanic and terrestrial subsurfaces, where the numbers of cells are 1.2 3 10 29 , 2.6 3 10 29 , 3.5 3 10 30 , and 0.25-2.5 3 10 30 , respectively. The numbers of het- erotrophic prokaryotes in the upper 200 m of the open ocean, the ocean below 200 m, and soil are consistent with average turnover times of 6-25 days, 0.8 yr, and 2.5 yr, respectively. Although subject to a great deal of uncertainty, the estimate for the average turnover time of prokaryotes in the subsurface is on the order of 1-2 3 10 3 yr. The cellular production rate for all prokaryotes on earth is estimated at 1.7 3 10 30 cellsyyr and is highest in the open ocean. The large population size and rapid growth of prokaryotes provides an enormous capacity for genetic diversity. Although invisible to the naked eye, prokaryotes are an essential component of the earth's biota. They catalyze unique and indispensable transformations in the biogeochemical cy- cles of the biosphere, produce important components of the earth's atmosphere, and represent a large portion of life's genetic diversity. Although the abundance of prokaryotes has been estimated indirectly (1, 2), the actual number of pro- karyotes and the total amount of their cellular carbon on earth have never been directly assessed. Presumably, prokaryotes' very ubiquity has discouraged investigators, because an esti- mation of the number of prokaryotes would seem to require endless cataloging of numerous habitats. To estimate the number and total carbon of prokaryotes on earth, several representative habitats were first examined. This analysis indicated that most of the prokaryotes reside in three large habitats: seawater, soil, and the sedimentysoil subsur- face. Although many other habitats contain dense populations, their numerical contribution to the total number of pro- karyotes is small. Thus, evaluating the total number and total carbon of prokaryotes on earth becomes a solvable problem. Aquatic Environments. Numerous estimates of cell density, volume, and carbon indicate that prokaryotes are ubiquitous in marine and fresh water (e.g., 3-5). Although a large range of cellular densities has been reported (10 4 -10 7 cellsyml), the

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Prokaryotes: The unseen majority

Evolution of Protein Molecules

Over three decades of molecular-phylogenetic studies, researchers have compiled an increasingly robust map of evolutionary diversification showing that the main diversity of life is microbial, distributed among three primary relatedness groups or domains: Archaea, Bacteria, and Eucarya. The general properties of representatives of the three domains indicate that the earliest life was based on inorganic nutrition and that photosynthesis and use of organic compounds for carbon and energy metabolism came comparatively later. The application of molecular-phylogenetic methods to study natural microbial ecosystems without the traditional requirement for cultivation has resulted in the discovery of many unexpected evolutionary lineages; members of some of these lineages are only distantly related to known organisms but are sufficiently abundant that they are likely to have impact on the chemistry of the biosphere.

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A Molecular View of Microbial Diversity and the Biosphere

After nearly 10 years of PCR-based analysis of prokaryotic small-subunit ribosomal RNAs for ecological studies it seems necessary to summarize reported pitfalls of this approach which will most likely lead to an erroneous description on the microbial diversity of a given habitat. The following article will cover specific aspects of sample collection, cell lysis, nucleic acid extraction, PCR amplification, separation of amplified DNA, application of nucleic probes and data analysis.

Determination of microbial diversity in environmental samples: pitfalls of PCR‐based rRNA analysis

A census of the biomass on Earth is key for understanding the structure and dynamics of the biosphere. However, a global, quantitative view of how the biomass of different taxa compare with one another is still lacking. Here, we assemble the overall biomass composition of the biosphere, establishing a census of the ≈550 gigatons of carbon (Gt C) of biomass distributed among all of the kingdoms of life. We find that the kingdoms of life concentrate at different locations on the planet; plants (≈450 Gt C, the dominant kingdom) are primarily terrestrial, whereas animals (≈2 Gt C) are mainly marine, and bacteria (≈70 Gt C) and archaea (≈7 Gt C) are predominantly located in deep subsurface environments. We show that terrestrial biomass is about two orders of magnitude higher than marine biomass and estimate a total of ≈6 Gt C of marine biota, doubling the previous estimated quantity. Our analysis reveals that the global marine biomass pyramid contains more consumers than producers, thus increasing the scope of previous observations on inverse food pyramids. Finally, we highlight that the mass of humans is an order of magnitude higher than that of all wild mammals combined and report the historical impact of humanity on the global biomass of prominent taxa, including mammals, fish, and plants.

https://www.pnas.org/content/pnas/115/25/6506.full.pdf

The biomass distribution on Earth

Intraterrestrial life has been found at depths of several thousand metres in deep sub-sea floor sediments and in the basement crust beneath the sediments. It has also been found at up to 2800-m depth in continental sedimentary rocks, 5300-m depth in igneous rock aquifers and in fluid inclusions in ancient salt deposits from salt mines. The biomass of these intraterrestrial organisms may be equal to the total weight of all marine and terrestrial plants. The intraterrestrial microbes generally seem to be active at very low but significant rates and several investigations indicate chemolithoautotrophs to form a chemosynthetic base. Hydrogen, methane and carbon dioxide gases are continuously generated in the interior of our planet and probably constitute sustainable sources of carbon and energy for deep intraterrestrial biosphere ecosystems. Several prospective research areas are foreseen to focus on the importance of microbial communities for metabolic processes such as anaerobic utilisation of hydrocarbons and anaerobic methane oxidation.

Exploration of deep intraterrestrial microbial life: current perspectives

Granitic rock has aquifers that run through faults and single or multiple fracture systems. They can orientate any way, vertically or horizontally and usually, only parts of hard rock fractures are water conducting. The remaining parts are filled by coatings of precipitated minerals, and clay and gouge material. Sampling hard rock is difficult and the risk of contamination due to intrusion of drilling fluids and cuttings in aquifers is obvious. A recent investigation of the potential for contamination of boreholes in granite during drilling operations, using molecular and growth methods, showed that predominating microorganisms in the drilling equipment were absent in groundwater from the drilled boreholes. The total number of bacteria found in subterranean granitic environments ranges from 103 up to 107 cells per ml groundwater, but the number of cultivable microorganisms is usually much lower. We have used culturing techniques with numeric taxonomy for the identification of cultivable microorganisms and the 16S rRNA gene technique to determine bacterial diversity in granitic groundwater. Members of the genera Bacillus, Desulfovibrio, Desulfomicrobium, Eubacterium, Methanomicrobium, Pseudomonas, Serratia and Shewanella have been found. Several biogeochemical processes in granitic rock have been demonstrated where microorganisms seem to be of major importance. One process is the mobilization of solid phase ferric iron oxy-hydroxides to liquid phase ferrous iron by iron reducing bacteria with organic carbon as electron donor. Another biogeochemical process found to be important is the reduction of sulfate to sulfide by sulfate reducing bacteria. They frequently appear in granitic aquifers at depths, and seem to prefer a moderate salinity, approximately 1%. When groundwater rich in ferrous iron, manganese(II) and reduced sulfur compounds reaches an oxygenated atmosphere such as an open tunnel, gradients suitable for chemolithotrophic bacteria develop. A third process is the conversion of carbon dioxide to organic material with hydrogen as the source of energy, possibly formed through radiolysis, mineral reactions or by volcanic activity. Recent results show that autotrophic methanogens, acetogenic bacteria and acetoclastic methanogens all are present and active in deep granitic rock. These observations announce the existence of a hydrogen driven deep biosphere in crystalline bedrock that is independent of photosynthesis. If this hypothesis is true, life may have been present and active deep down in the earth for a very long time, and it cannot be excluded that the place for the origin of life was a deep subterranean igneous rock environment (probably hot with a high pressure) rather than a surface environment.

Karsten Pedersen

Papers

Exploration of deep intraterrestrial microbial life: current perspectives

Microbial life in deep granitic rock

Biofilm development on stainless steel and PVC surfaces in drinking water

The environmental impact of mine wastes — Roles of microorganisms and their significance in treatment of mine wastes

The deep subterranean biosphere