Analysis | Published:

Bacteria and archaea on Earth and their abundance in biofilms

Nature Reviews Microbiologyvolume 17pages247260 (2019) | Download Citation

Abstract

Biofilms are a form of collective life with emergent properties that confer many advantages on their inhabitants, and they represent a much higher level of organization than single cells do. However, to date, no global analysis on biofilm abundance exists. We offer a critical discussion of the definition of biofilms and compile current estimates of global cell numbers in major microbial habitats, mindful of the associated uncertainty. Most bacteria and archaea on Earth (1.2 × 1030 cells) exist in the ‘big five’ habitats: deep oceanic subsurface (4 × 1029), upper oceanic sediment (5 × 1028), deep continental subsurface (3 × 1029), soil (3 × 1029) and oceans (1 × 1029). The remaining habitats, including groundwater, the atmosphere, the ocean surface microlayer, humans, animals and the phyllosphere, account for fewer cells by orders of magnitude. Biofilms dominate in all habitats on the surface of the Earth, except in the oceans, accounting for ~80% of bacterial and archaeal cells. In the deep subsurface, however, they cannot always be distinguished from single sessile cells; we estimate that 20–80% of cells in the subsurface exist as biofilms. Hence, overall, 40–80% of cells on Earth reside in biofilms. We conclude that biofilms drive all biogeochemical processes and represent the main way of active bacterial and archaeal life.

Introduction

There is a widely accepted but essentially unsubstantiated narrative that the majority of bacteria and archaea on Earth occur in biofilms1,2,3,4,5,6,7,8,9,10,11,12,13. Interestingly, most of these publications refer to a review paper by Costerton et al.1 from 1987, in which a generic reference is made to the ubiquity and importance of biofilms but without data supporting the claim that most archaea and bacteria on Earth live in biofilms. In this Analysis article, we probe the validity of the assumption that biofilms dominate globally by raising two fundamental questions: first, how many bacteria and archaea exist on Earth and where do they live? Second, what are biofilms and how many archaea and bacteria live in biofilms?

Both questions pose true challenges. There are few global estimates of bacteria and archaea on Earth, and they are based on local measurements that are then extrapolated. Current estimates point to the staggering number of ~1030 cells, with an approximate tenfold uncertainty14,15 (Fig. 1). This number is nine orders of magnitude larger than the currently known number of stars in galaxies in the universe (~1021)16. Here, we assess the number of cells living in biofilms in the largest abiotic and biotic habitats on Earth (assumptions and calculations provided in Table 1 and Supplementary Box 1) — notably of both bacteria and archaea, embracing archaea as biofilm-forming organisms17,18,19.

Fig. 1: Abundance of bacteria and archaea in different habitats on Earth.
Fig. 1

In total, ~1.2 × 1030 bacterial and archaeal cells exist on Earth, and the ‘big five’ habitats that bacteria and archaea inhabit are ocean, upper oceanic sediment (to 50 cm of depth), deep oceanic subsurface, soil (to 8 m of depth) and deep continental subsurface. All other habitats, including groundwater, waste water, animals and plants, each amount to 1027 or fewer cells (Table 1; Supplementary Box 1).

Table 1 Total and biofilm numbers of bacterial and archaeal cells in the most relevant habitats on Earth

Numbers range vastly, and estimates should be considered in terms of orders of magnitude. Nevertheless, there is a sizeable gap between the five largest habitats and all other microbial habitats. The ‘big five’ habitats are the deep oceanic subsurface, the deep continental subsurface, the upper oceanic sediment, the soil and the oceans (Fig. 1; Table 1). The deep continental subsurface and deep oceanic subsurface hold ~60% of all microbial cells in the entire biosphere of the Earth (Supplementary Box 1) at a ratio of 1:102 to 1:103 of free-living bacteria and archaea to surface-attached bacteria and archaea15,20,21.

Besides noting the uncertainty in numbers, one must also acknowledge that the definition of biofilms has been changing and broadening over time (Box 1). We suggest that biofilms should be defined according to their main characteristics (Box 2); however, not all of these characteristics necessarily apply to each and every biofilm. We also consider that there is a continuum between single sessile cells at one end and fully developed biofilms at the other end.

In the following sections, we consider the number of bacterial and archaeal cells that exist in the different environments found on Earth, and, mindful of the mentioned uncertainties, we estimate their abundance in biofilms and their global importance.

Deep suboceanic biosphere

The deep suboceanic biosphere includes nearly 70% of the surface of the Earth22, with an estimated volume of 1018 m3. It includes three main layers: a basalt layer that is 0.5–1 km thick, a layer of sheeted dike complex and a bottom layer of gabbroid rock in which 90% of the suboceanic biomass resides14. The basaltic layer is the main layer with substantial porosity and hence space for microbial habitation and processes22. On the basis of modelled 120 °C isotherms (assuming 120 °C as the temperature limit for microbial life), it has been estimated that the habitable deep suboceanic zone ranges from 0.5 km in the 1-million-year-old crust down to 5 km in the 180-million-year-old crust23. Crustal communities mediate the flux of crucial elements from the mantle to the overlying water24. The ocean crustal aquifer is the largest aquifer on Earth and encompasses 2% of the volume of the ocean25. It is by no means separated from the surface: the entire volume of the ocean is circulated through this aquifer every 70,000 years26. This results in a continuous exchange of water and solutes over geological times through the subocean aquifer system25. Tectonics also contributes to fluid movement by opening and closing fractures. In fact, deep seafloor sediments account for a large proportion of the global repository of microorganisms, and archaea constitute up to 35% of the biomass in marine subsurface sediments27,28 according to lipid analysis29,30, although the validity of this method has been questioned31.

Sampling and quantification of microbial biota in the deep biosphere remain extremely challenging, considering the various habitats in which cells can colonize fractured rocks (Fig. 2). A common method to assess the number of attached cells is to determine the number of planktonic cells in groundwater and multiply it by a factor of 102–103 according to a rough estimate of the attachment ratio20,21,32. Direct methods for quantification of sessile cells have been developed with homogenization of rock samples, followed by separation and enrichment of enclosed microbial cells, which are then counted using fluorescence microscopy33,34. However, neither the direct nor indirect methods can distinguish between single cells and microclusters or mature biofilms.

Fig. 2: Microbial habitats in fractured suboceanic and continental basaltic rock.
Fig. 2

Microbial cells usually grow in microclusters as epiliths on the surface of rocks, as chasmoendoliths in rock fissures, as cryptoendoliths in porous rock or as euendoliths by actively penetrating rock, apart from the few that occur as planktonic cells33; in glassic basalts, microbial activity by euendolithic organisms was demonstrated202.

In the deep ocean crust, mean cell numbers generally decrease from 1 × 108 to 5 × 108 cells per cm3 near the upper sediment surface to 106 cells per cm3 at the 1,000 m subsurface level; subseafloor sediments still contain 10–10,000-fold more cells per unit volume than productive ocean surface waters35,36. In a landmark study, Whitman and colleagues37 estimated the total cell number in subseafloor sediments to be as high as 4 × 1030 cells; subsequently, Kallmeyer et al.38 used a database of much greater geographical diversity, including gyre areas with extremely low cell abundance, and provided a lower estimate of 3 × 1029 cells. Microbial numbers in the deep ocean crust can vary between sites by five orders of magnitude depending on mean sedimentation rates and distance from the shore. A more recent estimate of 5 × 1029 cells also considers gas hydrate deposits and oil reservoirs39. Bar-On et al.14 took the geometric mean of those values and suggested 4 × 1029 as an estimate of the total cell number, with an approximate eightfold uncertainty.

The deep biosphere is highly dynamic and an integral part of biogeochemical fluxes and processes in the Earth's system over geological timescales39. Although activities are generally low, with generation times up to thousands of years, the overall activity calculations demonstrated that subsurface sediments can be responsible for the majority of sediment activity and hence are biogeochemically relevant on a global level40. By far the largest reservoir of methane on Earth is buried in marine sediments. It originates from both abiotic and biotic sources. The biotic proportion is formed from the reduction of carbon dioxide or low-molecular-weight organic compounds by the sessile microbiota in the subsurface41,42. Boetius et al.43 showed that a marine microbial consortium performed anaerobic oxidation of methane in anoxic sediments, most of it, arguably, in biofilms. In Miocene-aged coal beds 2 km below the sea floor, which are considered hot spots for microbial life, probing with a stable isotope detected methyl compounds fuelling slow growth in coal and shale beds44.

Over millions of years, cells can end up in the deep subsurface after slow burial in sediment layers40, where they contribute to sediment cohesion and diagenesis45. Many cells die, but a subset can survive46, for example, facultative anaerobes, fermenters and spore formers47. Cells can also exist in dormant states or have extremely low turnover rates35,39,48,49,50. Further, cells can be transported into deep layers by flowing through connected pores, fractured rocks or geological fault zones; there are regions of considerable fluid movement, which is driven by advection of seawater into the crustal materials at the sea floor and by thermal advection of geothermally heated waters47,51. Generally, subsurface locations with gaseous or liquid flow are the regions most likely to support microbial growth52.

Life in the Earth’s crust consists of microorganisms that support complete ecosystems using both lithoautotrophy and heterotrophy40. Thus, the ability of subsurface microorganisms to subsist in the energy-deprived deep biosphere is not acquired during burial, and their capacity to endure diverse stressors, including high pressure53,54,55, is characteristic of life processes in this unique environment. They are considered descendants of rare members of microbial communities in surface sediments that become predominant during burial56. Remaining organic material from upper sediments supports life in the deep sedimentary biosphere49. Basaltic rocks, as the major component of the upper kilometres of the igneous ocean crust, are rich in reduced chemicals such as iron and sulfur, which play a crucial role for microbial growth in these habitats51. A further energy source is hydrogen, a result of serpentinization in fluid–rock interactions57. Leftovers of plankton that were buried millions of years ago can still be degraded at very slow rates by microbial microconsortia that amount to millions of cells per cubic centimetre58. It has been shown that microorganisms can remain viable in deep ancient deposits at 250–320 m below the sea floor and survive on organic matter that was buried 7–11 million years earlier59. Culturable bacteria were isolated from almost 2,000 m below the sea floor of the Canterbury basin60.

Nitrogen is a possible limiting factor, as it is present in only small amounts (<100 µM) in seawater and in trace amounts in basalt51; however, the availability of energy sources might be the most important growth-limiting factor61. With stable isotope incubation and nanometre-scale secondary ion mass spectroscopy, it was confirmed that individual microbial cells from 460,000-year-old sediments 219 m deep in the southwestern Pacific incorporated 13C and 15N after uptake of exogenic glucose and pyruvate61. Ancient bacteria are capable of DNA repair, even if the cells are up to half a million years old62, and based on CARD–FISH, it was argued that bacteria with an active DNA-repair mechanism are the most likely to survive. In fact, a large fraction of subseafloor microorganisms has to be considered alive61, even in 16-million-year-old and deep (>400 m) sediments46. On the basis of CARD–FISH, the total number of living bacteria in the oceanic subsurface was estimated at 1.3 × 1029 cells46. As not all living bacteria may be detected by CARD–FISH, this is a minimal estimate. All detected living cells were bacteria, and they had an estimated turnover time of 0.3–22 years. Hence, cells retain the capacity for metabolic activities even when deeply buried for geologically relevant periods of time.

Adhesion to surfaces has been seen as a survival strategy under energy limitation63. A plausible explanation is that substrates such as fatty acids can stick to the surface of particles and thus accumulate locally63,64,65. Furthermore, associations of genetically identical cells increased with depth, which was taken as a sign of ongoing cell division. Biofilms have been directly demonstrated on the surfaces of seafloor-exposed basalt36. Not surprisingly, preferential growth occurred in crevices, pits and grooves on the surface of minerals66 (Fig. 2). Secondary mineral oxides and organic carbon can accumulate in such pits and fractures, leading to microniches that can be colonized by different microorganisms66.

The fast-growing microorganisms on the surface of the Earth affect elemental cycles on short timescales, but the energy-limited and extremely slow-growing majority of microorganisms strongly influence elemental cycles over geological periods64, representing a very large carbon sink.

Thus, it is confirmed that the deep marine biosphere is active and not just a reservoir of buried, inactive microbial cells47,66,67,68. We suggest that once the cells grow, even if ever so slowly, they will form clusters rather than move away, and it is plausible to classify the deep oceanic subsurface as a habitat dominated by biofilms, particularly the biogeochemically active regions. We estimate that 20–80%, or from 0.8 × 1029 to 3.2 × 1029 cells, exist in biofilms (Supplementary Box 1).

Deep continental subsurface

The deep continental subsurface has been defined as a depth from 8 m below the ground surface, excluding soil. It is considered habitable69 down to ~5,000 m below the ground surface14. The continental subsurface was reported to harbour ~2 × 1030 microbial cells, with an approximate 14-fold uncertainty14; however, in a very recent and comprehensive metastudy, Magnabosco et al.15 published a much lower range, from 2 × 1029 to 6 × 1029 cells, on the basis of data from more than 200 publications and ~3,800 cell concentration measurements. We have adopted the geometric mean of 3 × 1029 of this range for our analysis (Table 1; Supplementary Box 1).

These cells are of great relevance to life on the surface of the Earth because they are involved in all biogeochemical cycles70, similar to microbial communities in the oceanic subsurface. The large subcontinental biomass pool bridges the biological and geological element cycles71. In addition to photosynthesis driven by solar energy (and, in extreme cases, geothermal radiation72), chemical reactions within rocks providing ferric iron, geogenic hydrogen, methane and carbon dioxide are the major energy sources for life on Earth33,41. An entire autotrophic and heterotrophic biosphere has been proposed for the deep continental subsurface, in which hydrogen is used as the energy source for the reduction of carbon dioxide39,41,47. Detailed chemolithotrophic microbial loops, for example, based on iron, have been hypothesized39,41,47,73. Subcontinental microorganisms affect mineral formation and dissolution kinetics by altering the microenvironmental geochemistry, such as the pH value, redox potential, concentrations of dissolved salts and conductivity, and thus are actively involved in biogeochemical rock–water interactions73,74. An example is dissolution or ‘biological weathering’ of solid-phase ferric iron oxohydroxides by iron-reducing bacteria that use organic carbon as an electron donor75. Biogenic minerals range from carbonates, silicates, clays, iron and manganese oxides to sulfur and saltpetre at dimensions ranging from microscopic to macroscopic76,77,78. The organic carbon required for subcontinental microbial life can be generated by conversion of carbon dioxide with hydrogen as an electron donor, possibly formed by radiolysis, mineral reactions (for example, between crushed basalt and groundwater79), serpentinization57 or volcanic activity41,75,80.

Most subcontinental microorganisms are attached to surfaces and exceed the number of planktonic cells by several orders of magnitude. Whitman et al.37 estimated that only 0.06% of the cells in aquifers are unattached compared with 0.22% in groundwater wells. Finer-grained sediments have a higher ratio of attached to unattached cells32,81, with attached cells showing more activity than unattached cells20. McMahon and Parnell21 concluded that about 2.5 × 1029−2.5 × 1030 cells exist attached to subcontinental interfaces, far outnumbering planktonic cells (5 × 1027).

The existence of biofilms in the deep subsurface was confirmed for deep groundwater wells (down to 1,200 and 1,800 m), revealing massive biofilms in the granitic rock subsurface82,83. Bacteria from such wells easily formed biofilms in laboratory experiments78,84. Fossilized biofilms detected by transmission electron microscopy of drilling cores suggest the presence of biofilms in the subsurface for a very long time75. Scanning electron microscopy revealed that mineral surfaces 2.8 km deep were colonized by both dispersed individual cells and microcolonies; the average surface density was 5 × 104 bacteria per cm2, and the biofilm population exceeded that of the bacteria in the aqueous phase by two orders of magnitude85. Active microbial multispecies biofilms in deep, porous continental subsurface rocks were verified by CARD–FISH and a fluorescence lectin-binding assay (FLBA)86. All colonies exhibited traces of extracellular polymeric substances (EPS) surrounding them. The signals from EPS were not only concentrated in single colonies but also extended along the substrate matrix, connecting clusters86. Intercellular communication is expected in such clusters. The size of the ‘calling distance’ of quorum sensing has been shown to extend up to 78 µm in single-species biofilms87. However, in subsurface environments, in which confined space can limit the aggregation of cells, the possibility of communication and cooperation by diffusion of metabolites and quorum-sensing signals between different microcolonies, even at larger distances, should not be disregarded86. Hard rock interfaces provide space for specialized microniches with conditions favourable to different microorganisms and enable the interchange of metabolic products. This can generate a network of specialized metabolisms that would be impossible in a liquid-only world.

Subsurface microbial processes facilitated by biofilms are the basis for degradation and removal of contaminants along the underground passage of fluids and provide natural water purification and bioremediation. They keep groundwater reserves pristine not only by degrading organic matter but also by retaining metals that bind to biofilms88. Subsurface biofilms can strongly influence the porosity and permeability of porous media89; they act as a filter, reduce permeability and trap fine-grained particles and colloidal material90. Trapped minerals have much higher chemical and physical stability than the biofilm and can persist long after the biofilm has decayed or been removed89. Bacteria indigenous to granitic environments have been shown to strongly affect the hydrological regime77. This effect has to be considered when assessing the safety of subsurface storage of radioactive waste82,91 or sequestered carbon dioxide92.

In general, the existence of biofilms in the continental subsurface has been confirmed, and there is plenty of evidence that the cells are not simply buried and inactive but participate in global biogeochemical processes on geological timescales. We argue that these cells are the main driving force of geomicrobiological processes and estimate that 20–80%, or 0.6 × 1029–2.4 × 1029 cells, exist in biofilms (Supplementary Box 1).

Upper ocean sediments

The uppermost centimetres represent the biologically most active sedimentary layer93,94, even in the deepest trenches of the oceans and regardless of the height of the water column above it95. Continental shelf sediments are mostly anoxic immediately below the sea floor47,96, but 9–37% of global subseafloor sediments are oxygenated and harbour aerobic microorganisms97. The deposition of marine snow, detritus and biodegradable particles provides ample nutrients for sediment microorganisms. Their density was reported as between 106 and 108 cells per cm3 of sand; however, only a small portion of the available particle surface is colonized — between 0.01% and 5% depending on the cell detection method14,93,98. The number of attached cells exceeds that of unattached cells in sediments and aquifers by a factor of 102–103 owing to the high cell densities of attached aggregates21,32,38,81,99. Therefore, the number of free-living, planktonic cells in sediments can be neglected compared with the number of attached cells.

In an early seminal study, Weise and Rheinheimer100 investigated the colonization of sand grains and found many patches of aggregated microorganisms. The average microbial density was 108 per cm3. The microtopography of the grains often predetermined the locations with the highest population and detritus density, namely, surface fissures, surface steps, slight indentations and conchoidal breakage sites. Bacteria can act as glue for minute particles, which increases the size of sedimentary particles. In fact, biofilms play an important role in stabilizing sediments45,101,102 and the subsequent diagenesis103,104. This suggests that biofilms strongly influence sediment texture and stability. In such an environment, cell clusters and colonies on mineral and detritus surfaces are prevalent biofilm manifestations.

To summarize, the vast majority of cells in sediments down to 50 cm depth exist as biofilms, however, at much lower numbers than originally published by Whitman et al.37. Bacteria are now estimated at 4 × 1028 cells and archaea at 1 × 1028 cells28 in the upper ocean sediment, with archaeal numbers decreasing more slowly with depth than bacterial numbers27.

Soil

Soil is the most heterogeneous component of the biosphere in terms of properties and processes and offers a huge internal surface area105. Soil contains high numbers of bacteria and archaea, mainly in the form of microbial aggregates106, with estimated total cell numbers ranging from 1 × 1027 for tropical rainforests to 6.3 × 1028 for desert scrub and a total of 3 × 1029 cells37. This number was confirmed by Bar-On et al.14 at an uncertainty of fourfold for bacteria and sixfold for archaea14,107.

Soil presents environments that vastly differ in their physical, chemical and biological properties108,109,110. Stable microaggregates and macroaggregates of soil material have been demonstrated by physical fractionation, harbouring different microbial populations; bacteria have been reported to be less prone to protozoal predation if attached to clay owing to the protective effect of their EPS108,111,112. Microorganisms are embedded in soil-solute films of 2–10 µm thickness, adhering to surfaces of pores110. Biofilms fundamentally change the physicochemical characteristics of soil pores and their dimensions. They cause local gradients in nutrient level, oxygen concentration, redox potential and pH value. They lead to local assembly of biological materials, which can change surface properties, water retention and flow. EPS in soil increase heterogeneity113 and improve aggregate stability114. The quorum size for biofilm-specific activities in environments in which diffusion rates are low can be small because there is little signal loss. Under these conditions, the number of cells needed to communicate by quorum sensing can be as low as 30–50 cells, well within the number of neighbours that bacterial cells have in soil110. On the basis of the very high density of attached cells in soil and their metabolic activity, we conclude that soil bacteria and archaea live in biofilms, encompassing roughly 3 × 1029 cells.

Oceans

Surface microlayer and neuston

The surface microlayer (SML) is the interface between water and the atmosphere, comprising the first millimetre of the water phase115. It covers roughly 70% of the planetary surface, is involved in heat, momentum and mass exchange between the atmosphere and the hydrosphere and has been called 'the ocean’s vital skin' (ref.116). The organisms living in the SML are referred to as neuston (as opposed to nekton or plankton, living at greater depths) and include a range of diverse microorganisms adapted to this environment117,118. The cell density of the ‘bacterioneuston’ is 3–5 orders of magnitude higher than that of cells in the bulk water phase119.

Microbial cells in the SML occur as aggregates, and the SML can be considered a gelatinous biofilm120, although mixing by wind and waves may prevent long-term stability of the bacterioneuston community121,122. All the gas exchange between water and the atmosphere happens across the SML123 and is heavily influenced by biosurfactants produced by neuston organisms, as well as by hydrophobic organic and metal compounds124, which reduce the water surface tension125. The SML is a highly dynamic and stressful environment owing to large temperature and salinity changes, high ultraviolet radiation and accumulated metals, hydrocarbons and hydrophobic particles. Both release and deposition of aerosolized microorganisms involve the SML117.

Given a global water surface of 361 × 106 km2, the volume of the first millimetre is 361 km3. Microbial cell concentrations in the neuston126 are of the order of 105–106 cells per millilitre (ref.1); hence, total numbers range from 0.4 × 1023 to 4 × 1023 cells. This makes the neuston a minor contributor to the overall aggregated-cell repository despite its high cell density and global relevance.

Open ocean water

It is commonly thought that bacteria in the open ocean occur as single cells and that there are ~1 × 1029 microbial cells14,27,37,127,128,129, 20% of which are archaea14. Compared with the sea, fresh water (rivers and lakes) covers a much smaller area, and the freshwater cell numbers can be neglected here14.

The aerobic, heterotrophic SAR11 clade and the phototrophs Prochlorococcus and Synechococcus contribute a substantial fraction to marine primary production, with annual mean global abundances of 2 × 1028 cells for SAR11 (refs130,131) and 3 × 1027 and 7 × 1026 cells for Prochlorococcus and Synechococcus, respectively132. SAR11 cells are minimal in size and complexity, a phenomenon called streamlining133, suitable for lowering the cost of replication and maximizing transport functions, which is optimal for competition at extremely low nutrient availability. They are photochemotrophs and self-sufficient; their small body size provides a large surface area, improving nutrient adsorption.

SAR11 has been overwhelmingly observed as single cells and clearly has to be considered planktonic134. Interestingly, Prochlorococcus cannot be cultivated as a pure culture in its own natural habitat (sunlight and seawater), because it soon becomes poisoned by hydrogen peroxide, which is produced by photo-oxidation135. Although Prochlorococcus ancestors exhibited catalase–peroxidase, this function was lost and has been replaced by commensal growth with Synechococcus, a catalase–peroxidase-producing species135. Physical colocalization might be hypothesized under these conditions, although the formation of aggregates has not (yet) been reported. Although Synechococcus has been found in periphytic biofilms in freshwater environments136, available data suggest that biofilms are not a major way of life for SAR11, Synechococcus or Prochlorococcus in the open ocean.

The case for biofilms in the oceans

It is debatable whether all microorganisms in the oceans occur as single cells — common sampling techniques mostly do not distinguish between planktonic and aggregated cells14. In the few studies that differentiate between planktonic cells and aggregates, elaborate sampling was required, usually by serial filtration through filters of decreasing pore size137, glass fibre filters138,139 or cellulose ester filters140,141. These studies reported many aggregates that qualify for the general definition of biofilms, but a quantitative comparison with single cells was not attempted. Abiotic and biotic particles offer attachment surfaces and are usually heavily colonized139,142,143. Cells attached to macroaggregates and microaggregates comprised 3% and 4%, respectively, of the overall cell number14. Particle-attached bacteria tend to be larger, with a higher proportion of cells with higher metabolic activity, than free-living bacteria144, although their abundance varies with location and season. Under eutrophic situations when algal blooms occur, a huge proportion of bacteria are attached to phytoplankton and zooplankton as epibiotic biofilms65,145,146, as plankton in eutrophic waters represent a nutrient hot spot for heterotrophic bacteria. Mineral particles are also highly colonized139. Accumulating plastic debris (plastisphere) in the oceans142,143,147,148 represents an emerging source of surfaces for microbial attachment. The available surface area is continuously increasing as the abrasion between plastic particles generates ever more microparticles. Modelling suggests that plastic particles <10 µm end up in the sediment owing to colonization by microorganisms and the resulting increase in density149. The prospects for complete elimination by biodegradation are dim150; instead, degradation processes will fragment the particles and increase the overall surface area. Estimates regarding the overall surface area of the plastisphere are not available but must be high151. The microbial load travelling with the particles has been identified as a potential health hazard. For example, there is evidence for facultative pathogenic Vibrio spp. hitch-hiking on microplastic particles152.

A well-known phenomenon of aquatic microbial aggregates is marine, lake and river snow153,154,155,156. These aggregates of organic matter are abundant and are sites of microbial activities, including intense intercellular signalling157. Marine snow is densely populated by multispecies microbial aggregates, which have been shown to be taxonomically and genomically distinct from planktonic microorganisms158,159.

Transparent exopolymer particles (TEP) are defined as colloidal material that can be visualized by staining with Coomassie blue118,153 and usually contain embedded microorganisms160,161. These highly hydrated polymers consist mainly of polysaccharides and are scaffolds for microorganisms, analogous to the EPS matrix in other biofilms162.

To conclude, although the majority of microorganisms (~1029 cells) in the ocean seems to exist as single cells, the global fraction of cells bound to mineral and microplastic particles163, to organic debris, to phytoplankton and zooplankton, to macrophytes such as kelp146 and to corals and sponges is unknown (Fig. 3). Thus, the case for biofilms in oceans is still open.

Fig. 3: Interfaces colonized by biofilms in the oceans.
Fig. 3

Although oceans are considered to host mostly planktonic microbial cells, zooplankton and phytoplankton, abiotic minerals (not shown), organic and synthetic (plastic) particles, macrophytes, corals and sponges provide huge surface areas for microbial biofilms. TEP, transparent exopolymer particles.

Atmosphere

The atmosphere contains substantial numbers of microorganisms164. Their distribution varies greatly, depending on altitude, location (whether over land, particularly agricultural, open sea or coastal areas) and wind forces, with extremely high particle concentrations in hurricanes, tornadoes and cyclones164. Aerosols can originate from any surface, including soil, water and plants, and carry microorganisms to the atmosphere165. Bursting of small bubbles on wave caps continuously transfers bacteria from sea to air. The bubbles pass the SML, which contains high concentrations of microorganisms and organic matter (see above), capturing this material, and form microaerosols, which are enriched in bacteria by a factor of up to 2,500 (ref.166).

Some bacteria and fungi potentially affect cloud formation and precipitation because they function as nuclei for water condensation and ice formation; in the latter case, they are active at higher temperatures compared with inorganic nuclei167,168. Thus, they are intimately involved in global climate processes169. Once airborne, bacteria continue to be metabolically active in cloud droplets and influence droplet chemistry170,171, even at supercooled temperatures172. They can grow in cloud water, and doubling times between 3.6 days and 19.5 days have been reported172,173. Taxa such as Afipia sp., Oxalobacteraceae and Methylobacteriaceae, which can use C1–C4 compounds that are ubiquitously present in the atmosphere and cloud water, are frequently found in clouds164.

Microbial aerosols, composed of bacterial and fungal cells and spores, are considered to constitute up to 74% of the total aerosol volume168. Bacteria, bacterial spores and fungal spores have been detected at altitudes of up to 41 km and 78 km, respectively, with only occasional detection of cells at such altitudes174,175; most airborne microorganisms are found in the troposphere below an altitude of 11 km.

Mean concentrations in ambient air are estimated at 104 cells m−3, although the number may increase dramatically with strong winds164. Assuming a troposphere volume of 5 × 1018 m3, the total air microbiome amounts to roughly 5 × 1022 cells (Supplementary Box 1), and the number in cloud droplets alone is estimated at 1019 cells170. If bacteria are metabolically active in clouds170,176, it is conceivable that they grow and form aggregates. However, for this Analysis, we consider it unlikely that such aggregates constitute a substantial proportion of microorganisms in clouds.

Microbial communities of eukaryotes

The phyllosphere

Plant leaves constitute a huge microbial habitat (Fig. 4). Together with all the other above-ground parts of the plant, they form the phyllosphere. Biofilm-forming bacteria colonizing the surface of all plants177 were long ignored, and first acknowledged only in 1961 (ref.178).

Fig. 4: Biofilms in eukaryotic habitats.
Fig. 4

Plant leaves, humans, cattle and termites are colonized by microorganisms. These host-associated communities, ranging from the phyllosphere and rhizosphere to the gut microbiota, exhibit many of the key biofilm characteristics (Box 2).

Plant surfaces are stressful and unstable environments: they are oligotrophic in terms of easily available carbon and nitrogen sources and have multiple and highly fluctuating physicochemical stressors, such as light, ultraviolet radiation, temperature and water activity179. Preferential sites for colonization are epidermic cell wall junctions, glandular and nonglandular trichomes, veins and stomata180. Most bacteria are found in aggregates of at least 1,000 cells, and aggregate size positively correlates with water availability181.

The microbial communities on plant leaves are highly diverse, comprising bacteria, archaea, eukaryotes and viruses, which interact with one another and with the plant. There is a large body of evidence showing that phyllosphere microbial communities influence plant fitness and ecosystem functions182,183.

Average bacterial numbers on leaves184 range from 106 to 107 per cm2. The total surface area of the phyllosphere on land has been estimated to be between 5 × 108 and 6.4 × 108 square kilometres184, which is five times the land surface. Another estimate of 109 km2 considered both upper and lower leaf surface181. On the basis of this number, the global bacterial population in the phyllosphere comprises between 0.6 × 1026 and 6 × 1026 cells, with a geometric mean of 2 × 1026 cells (Supplementary Box 1).

The microbial community of the rhizosphere might represent a similar order of magnitude; however, we found no data to assess its total number. The same is true for aquatic plants, which are usually strongly colonized (with the notable exception of aquatic plants such as Delisea pulchra, which produce quorum-sensing inhibitors185). We assume that these habitats together harbour a community roughly equal to that of the phyllosphere. Still, plant-associated microorganisms do not reach the magnitude of the big five.

Humans

The gut microbiota is part of the human holobiont and interacts continuously with the host; it is structured like and fulfils some of the criteria of a biofilm, although perhaps not in a classical sense. In fact, the environment of cells in the gut186 meets most of the biofilm criteria given in Box 2.

It was long thought that there are more microbial cells than eukaryotic cells in humans and that the global human population might harbour a substantial proportion of the microorganisms on Earth. More recently, Sender et al.187 scrutinized the cell numbers of the human holobiont and found an approximate ratio of only 1:1 of bacterial to human cells, with the earlier assessment having been based on a string of citations dating back to one elegant, but inaccurate, back-of-the-envelope estimate188. Depending on the underlying estimates used for the number of gut microorganisms per millilitre (ref.37,187), the total number of gut bacteria of today’s human population is between 3 × 1023 and 5 × 1023 cells (Table 1; Supplementary Box 1); we used the geometric mean of 4 × 1023 cells for subsequent calculations. Dental plaque (8 × 1021 cells) and skin (1 × 1021 cells) are minor contributors of total bacterial cell numbers187. All these human habitats provide interfaces for biofilms (Fig. 4), although the total number of human microbiota cells is low compared with the big five habitats. Human and industrial waste waters contain ~1026 cells on an annual basis (Supplementary Box 1, based on previous work189,190) and were not considered in the calculations.

Livestock animals: cattle, pigs and poultry

We estimated microbial populations in cattle, pigs and domestic birds on the basis of global head counts available from the Food and Agriculture Organization of the United Nations (Table 1) and used these to compute their contributions to biofilms at a global level (Supplementary Box 1). Their combined number is of the order of 1024, with cattle contributing the most to this total.

Termites

Represent a very large population, harbouring many bacteria and archaea37,191,192 (Fig. 4). With a global termite population of 2.4 × 1017 and 2.7 × 106 bacteria and archaea per animal, the total number of bacterial and archaeal cells in the termite gut amounts to 6 × 1023 (Table 1).

Conclusions

Although the numbers compiled here are only crude estimates, they clearly reveal that the upper oceanic sediment, deep ocean subsurface, soil, deep continental subsurface and oceans represent the main habitats of bacteria and archaea on Earth. Assuming a ratio between attached and nonattached cells of 1,000:1, nonattached cells in pore and groundwater do not substantially influence the total numbers, except in the oceans. Cells in the upper sediment and in soil can be categorized as living in biofilms, and there is evidence for the existence and likely prevalence of biofilms in both the deep oceanic and continental subsurfaces. Such biofilms have an important role in the fate of geochemically relevant elements by influencing their sequestration for millions of years or their return to the surface, which affects life and climate. In oceans, most microorganisms probably exist, metaphorically speaking, as ‘professional planktonics’; however, the proportion of cells colonizing the huge surfaces of debris, plankton, aquatic plants and mineral and plastic particles as well as in TEP and marine snow is currently unknown and should be quantified — these cells would also qualify as biofilms. Other substantial niches of biofilms such as microbial mats and the surfaces of aquatic plants and animals also await quantification.

In summary, our estimate puts the total number of bacterial and archaeal cells on Earth at around 1.2 × 1030 cells. The answer to the abundance of biofilms is complex. Approximately 40% of all bacteria and archaea occur above the subsurface, 80% of them in soil and upper oceanic sediment as biofilms and 20% in oceans as planktonic cells. The majority of all cells reside in the continental subsurface and oceanic subsurface, with over 99% surface attached. If cells are active, sooner or later, they will divide and eventually form clusters and thus biofilms. Metabolic and microscopic evidence actually reveals that they form biofilms, even at very low growth rates, representing 20–80% of the overall cell number. We posit that the biogeochemical processes in the subsurface are generally driven by biofilms. This census outlines the global relevance of biofilms that shape life on Earth.

Additional information

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Acknowledgements

Many colleagues responded to requests for bacterial numbers or were ready to discuss them, which was highly appreciated: A. Boetius, R. Colwell, A. Decho, R. Gerlach, R. Glud, B. B. Jørgensen, K. Kjeldsen, S. Kjelleberg, F. Lauro, H. Lesch, R. Meckenstock, L. Melo, L. A. Meyer-Reil, J. Parkes, K. Pedersen, H. Peter, P. Rettberg, B. Schink, U. Schreiber, S. Schuster, P. Stoodley, W. Streit, S. Swarup, U. Szewzyk, M. Vera, G. Wolfaardt, O. Wurl and, above all, J. Wingender. Special thanks to K. Peter for help with figure drafts. Furthermore, the authors are very thankful to the reviewers whose thorough work helped to improve this Analysis.

Reviewer information

Nature Reviews Microbiology thanks Y. M. Bar-On, R. Milo, P. Stoodley and I. Wagner-Döbler for their contribution to the peer review of this work.

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Affiliations

  1. Singapore Centre for Environmental Life Sciences Engineering (SCELSE), Singapore, Singapore

    • Hans-Curt Flemming
    •  & Stefan Wuertz
  2. Biofilm Centre, Faculty of Chemistry, University of Duisburg-Essen, Essen, Germany

    • Hans-Curt Flemming
  3. School of Civil and Environmental Engineering, Nanyang Technological University, Singapore, Singapore

    • Stefan Wuertz

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  2. Search for Stefan Wuertz in:

Contributions

H.-C.F. did the data research and drafted the manuscript and figures; S.W. provided the idea to this work, substantially contributed to discussion of content and performed and edited the calculations.

Competing interests

The authors declare no conflicts of interest.

Corresponding authors

Correspondence to Hans-Curt Flemming or Stefan Wuertz.

Supplementary information

Glossary

Basalt

The most common type of solidified lava; a fine-grained igneous rock.

Dike

A rock sheet formed in crevices and fractures of an already existing rock body.

Gabbroid rock

A compact, dark, coarse-grained magmatic rock, chemically equivalent to basalt, that forms when molten magma is trapped in the subsurface, slowly cools and forms a crystalline mass.

Gyre

A large system of circulating ocean currents, caused by the Coriolis effect, involved with large wind movements. The five most notable gyres are the Indian Ocean gyre, the North Atlantic gyre, the North Pacific gyre, the South Atlantic gyre and the South Pacific gyre.

Emergent properties

The characteristics of a community not identifiable by analysing the component organisms in isolation, including novel and coherent structures, patterns and properties arising during the process of self-organization in complex systems — the whole is more than the sum of its parts.

Substratum

A solid surface on which organisms adhere and grow.

Diagenesis

All processes that happen during the transformation of a sediment to its final lithification. It is a low-pressure, low-temperature process that can involve microbial biofilms, owing to their extracellular polymeric substances, as opposed to metamorphism, a rock alteration process that occurs at high temperatures and pressures.

Igneous

A type of crystalline rock that forms directly from the cooling of magma.

Serpentinization

The hydrothermal transformation of primary ferromagnesian minerals producing fluids rich in hydrogen and various secondary minerals. The hydrogen can reduce carbon dioxide and initiate an inorganic pathway for organic compounds.

Canterbury basin

The sedimentary basin around the South Island of New Zealand.

Stable isotope incubation

The exposure of microbial communities to stable isotopes (for example, 13C- or 15N-labelled glucose, pyruvate and amino acids) to determine the incorporation and thus the metabolic activity of microorganisms.

Nanometre-scale secondary ion mass spectroscopy

(Nano-SIMS). A type of imaging with secondary ion mass spectroscopy with nanoscopic-scale resolution.

CARD–FISH

Fluorescence in situ hybridization (FISH) with horseradish-peroxidase-labelled oligonucleotide probes and tyramide signal amplification, also known as catalysed reporter deposition (CARD).

Chasmoendoliths

Organisms growing in fissures of rocks.

Cryptoendoliths

Organisms growing in deep cavities or crevices within rock.

Euendoliths

Organisms growing in cracks and pits actively penetrating the mineral material.

Extracellular polymeric substances

(EPS). Mainly polysaccharides, proteins, nucleic acids and lipids; they provide the mechanical stability of biofilms, mediate adhesion to surfaces and form a cohesive, 3D polymer network that interconnects and transiently immobilizes biofilm cells. In addition, the biofilm matrix functions as an external digestive system by retaining extracellular enzymes in close proximity to the cells that solubilize colloidal and solid biopolymers and thus make them bioavailable.

Quorum sensing

The sensing of microbial population density. This mechanism can regulate gene expression in response to fluctuations of cell-population density. It is based on the production and release of small soluble molecules named ‘autoinducers’ because they act not only on other cells but also on the producing ones once a threshold concentration is reached.

Conchoidal breakage sites

The locations of breakages that are characteristic of the way in which brittle materials break or fracture if they do not follow any natural planes of separation. Quartz, flint, quartzite, jasper and other fine-grained or amorphous materials, such as pure silica, obsidian and window glass, are among the materials that break in this way.

Plastisphere

The microbial community of heterotrophs, autotrophs, predators and symbionts living on plastic debris in oceans, fresh water, soils and sediments.

Troposphere

The lowest and densest part of the atmosphere, which extends up to ~11 km in altitude. It is where most of the weather changes occur and where the vast majority of microbial and abiotic aerosols are found.

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