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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 cycles 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 prokaryotes and the total amount of their cellular carbon on earth have never been directly assessed. Presumably, prokaryotes' very ubiquity has discouraged investigators, because an estimation 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 sediment/soil subsurface. Although many other habitats contain dense populations, their numerical contribution to the total number of prokaryotes 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 cells/ml), the mean values for different aquatic habitats are surprisingly similar. For the continental shelf and the upper 200 m of the open ocean, the cellular density is about 5 × 10 ⁵ cells/ml. A portion of these cells are the autotrophic marine cyanobacteria and Prochlorococcus spp., which have an average cellular density of 4 × 10 ⁴ cells/ml ( 6 ). The deep (>200 m) oceanic water contains 5 × 10 ⁴ cells/ml on average. From global estimates of volume, the upper 200 m of the ocean contains a total of 3.6 × 10 ²⁸ cells, of which 2.9 × 10 ²⁷ cells are autotrophs, whereas ocean water below 200 m contains 6.5 × 10 ²⁸ cells (Table 1 ).

In the polar regions, a relatively dense community of algae and prokaryotes forms at the water-ice interface in annual sea ice ( 11 ). In Antarctic sea ice, the estimated number of prokaryotes (2.2 × 10 ²⁴ cells) was based on the mean cell numbers of Delille and Rosiers ( 12 ) and the mean areal extent of seasonal ice ( 13 ). If the population size in the Arctic is similar ( 14 ), the global estimate for both polar regions is 4 × 10 ²⁴ cells, only a fraction of the total number of prokaryotes.

Soil. Soil is a major reservoir of organic carbon on earth and an important habitat for prokaryotes. Prokaryotes are an essential component of the soil decomposition subsystem, in which plant and animal residues are degraded into organic matter and nutrients are released into food webs ( 15 ). Many studies indicate that the number of prokaryotes in forest soils is much less than the number in other soils. The total number of prokaryotes in forest soil was estimated from detailed direct counts from a coniferous forest ultisol ( 16 ), which were considered representative of forest soils in general (Table 2 ). For other soils, including grasslands and cultivated soils, the numbers of prokaryotes appear about the same, e.g., the number of prokaryotes in Negev desert soil is comparable to the number in cultivated soil ( 19 ). Therefore, the numbers of prokaryotes in all other soils were estimated from the unpublished field studies of E. A. Paul for cultivated soils (cited in ref. 18 ).

Subsurface. The subsurface is defined here as terrestrial habitats below 8 m and marine sediments below 10 cm. Few direct enumerations of subsurface prokaryotes have been made, largely because of the difficulty in obtaining uncontaminated samples. Nevertheless, circumstantial evidence suggests that the subsurface biomass of prokaryotes is enormous ( 20 ). For instance, groundwater from deep aquifers and formation water from petroleum deposits contain 10 ³ -10 ⁶ prokaryotic cells/ml ( 21 , 22 ).

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Alternatively, the number of terrestrial subsurface prokaryotes can be estimated from groundwater data. Based on values from seven sites and four studies ( 31 , 37-39 ), the average number of unattached cells in groundwater is 1.54 × 10 ⁵ cells/ml. The total volume of groundwater in the upper 4 km of the earth's surface is 9.5 × 10 ²¹ cm ³ ( 40 ), and thus the number of unattached prokaryotes in groundwater is 1.46 × 10 ²⁷ cells. However, the number of prokaryotes in aquifer sediments is probably many orders of magnitude greater than the number unattached in the groundwater per se. For an aquifer 30-200 m deep, only 0.058% of the prokaryotes are unattached (calculated from the data of refs. 31 , 41 , and 42 ). This value appears to be representative of groundwater from other deep aquifers ( 22 , 37 ), which implies that the terrestrial subsurface contains about 2.5 × 10 ³⁰ prokaryotic cells. This estimate contains two major uncertainties. First, about 55% of the earth's groundwater is found below 750 m ( 40 ), and the extrapolation of values from the groundwater and aquifers above 750 m may not be applicable. Second, the ratio of unattached prokaryotes in aquifers was calculated from unconsolidated sediments, and the ratio may vary in other types of aquifers where the physical properties of the rocks and sediments are very different.

Other Habitats. Although they were found not to constitute a large fraction of the total number of prokaryotes, other habitats are of interest in their own right.

Although the number of prokaryotes in the gastrointestinal tracts of animals is enormous, it is unlikely to represent a large fraction of the total prokaryotes on earth. For example, the number of prokaryotes in the bovine rumen is 4-6 orders of magnitude less than the numbers found in soil, the subsurface, and sea water. Therefore, although the numbers of prokaryotes are known for only a few groups of animals, it is unlikely that animals contain a major fraction of the total number of prokaryotes.

Air. By volume, the atmosphere represents the largest compartment of the biosphere, and prokaryotes have been detected at altitudes as high as 57-77 km ( 66 ). Nevertheless, the total number of airborne prokaryotes appears to be quite low. For the bottom 3 km of the atmosphere, the total number of prokaryotes over land is about 5 × 10 ¹⁹ cfu (calculated from refs. 67-69 ), a value so low that it is unlikely that airborne prokaryotes represent a large fraction of the total number of prokaryotes.

Carbon Content. The amount of carbon in prokaryotes can be estimated from the cell numbers in soil, aquatic systems, and the subsurface. In the soil and subsurface, the cellular carbon is assumed to be one-half of the dry weight. In soil, the average dry weight of a prokaryotic cell is 2 × 10 ¹³ g or 200 fg ( 18 ). Thus, the total prokaryotic cellular carbon in soil is 26 × 10 ¹⁵ g of C or 26 Pg of C (Table 5 ). In the subsurface, there is only one measurement of the average dry weight of cells, that of 172 fg for cells from a terrestrial aquifer ( 36 ). This value yields an estimate of the terrestrial prokaryotic cellular carbon of 22-215 Pg of C (Table 5 ). The estimate for the marine subsurface, 303 Pg of C (Table 5 ), may be compared with 56 Pg of C, the value obtained by Parkes et al. ( 33 ). The difference, 5.4-fold, is due in part to how the depth integrations were calculated. Parkes et al. ( 33 ) used logarithmic extrapolations rather than arithmetic averages, which decreased their estimated number of cells by 3-fold. They also estimated the amount of carbon per cell at 65 fg of C rather than the 86 fg of C used here. The remaining difference occurs because the current estimate is based in part on additional marine and terrestrial data.

Discussion. The total carbon of prokaryotes on earth is enormous, approximately 60-100% of the total carbon found in plants (Tables 5 and 6 ). Inclusion of this carbon in global models will greatly increase estimates of the amount of carbon stored in living organisms. In addition, prokaryotes contain large amounts of N, P, and other essential nutrients. For instance, assuming a C/N/P ratio in prokaryotes of 1:0.24:0.025 ( 74 ), the entire prokaryotic pool for N and P is 85-130 Pg of N and 9-14 Pg of P. In all plants, assuming C/N and C/P ratios for the 471 Pg of plant C in forests and woodlands of 156 and 1340, respectively, and C/N and C/P ratios for the 88 Pg of plant C in other ecosystems of 12.5 and 125, respectively ( 73 ), the amounts of N and P are 10 Pg and 1.05 Pg, respectively. Thus, the plant pool for these nutrients is an order of magnitude smaller than the total prokaryotic pool. In fact, the amount of N and P in soil prokaryotes, 6.2 Pg and 0.65 Pg, respectively, is nearly equal to the amount in terrestrial plants even though terrestrial plants contain much more carbon. Other essential nutrients are probably distributed similarly, and prokaryotes may represent the largest living reservoir for these elements on earth.

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Results from a similar analysis for the subsurface prokaryotes are problematic. Assuming that 1 Pg of C/yr, or about 1% of the total net productivity, reaches the subsurface and that the net burial rate is 0.06 Pg of C/yr ( 73 ), only 0.94 Pg of C/yr is available to support the subsurface community of prokaryotes. If the efficiency of carbon assimilation is 0.20, then the calculated average turnover time is 1-2 × 10 ³ yr, far longer than found in other ecosystems. At present, a number of plausible explanations for this apparent anomaly exist. ( i ) The average turnover time could be on the order of 1,000 yr. If this were the case, most of the subsurface prokaryotes must be metabolically inactive and probably nonviable. Circumstantial evidence suggests that this is not the case, and viability of subsurface prokaryotes is within the range observed for prokaryotes from surface sediments and soils (cf. 24, 31). Sulfate reduction, methanogenesis, and other activities have also been detected in cores from the subsurface ( 24 ). Thus, although it is likely that the relative metabolic activity and rate of carbon consumption of subsurface bacteria are lower than that found on the surface, activity must still be sufficient to maintain culture viability. ( ii ) Lithoautotrophic processes may provide an additional source of energy for growth of subsurface prokaryotes. Although lithoautotrophy has been demonstrated in some geological formations, current evidence suggests that most of the subsurface biomass is supported by organic matter deposited from the surface ( 80-82 ). Because the data are so limited, future studies could revise this view. ( iii ) The subsurface biomass may be overestimated. The estimate of subsurface carbon is based on a conversion factor derived from data at one site, which may not be representative. However, given that some of the smallest cells so far described in nature contain 5 fg of C, the magnitude of this error is unlikely to be more than 10- to 20-fold. ( iv ) The efficiency of carbon assimilation may be underestimated. Pure culture studies with rich media suggest that the efficiency of carbon assimilation can be as high as 0.85 ( 83 ). However, the error associated with this factor cannot be more than 4-fold. These points, when considered together, emphasize that our current understanding of subsurface prokaryotes is incomplete. Because of their numerical importance, more extensive examination of this habitat is imperative.

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For essentially asexual, haploid organisms such as prokaryotes, mutations are a major source of genetic diversity and one of the essential factors in the formation of novel species. Given prokaryotes' enormous potential to acquire genetic diversity, the number of prokaryotic species may be very large. Recent estimates for the number of prokaryotic species range from 10 ⁵ to 10 ⁷ ( 88 ). However, the current definition of a prokaryotic species, which includes strains whose genomic DNAs form hybrids with a change in the melting temperature ( T _m ) of less than 5°C ( 89 ), may be misleading. Application of the same definition to eukaryotes would lead to the inclusion of members of many taxonomic tribes into the same species ( 90 ). Similarly, phylogenetic groups such as humans, orangutans and gibbons would also belong to the same species ( 91 ). Thus, a simple comparison of the number of eukaryotic and prokaryotic species greatly underestimates prokaryotic diversity. Given prokaryotes' numerical abundance and importance in biogeochemical transformations, the absence of detailed knowledge of prokaryotic diversity is a major omission in our knowledge of life on earth.