Recent studies confirm that bacteria exist in the stratosphere. It is generally assumed that these bacteria are exiting from Earth, although it is possible that some are incoming from space. Most stratospheric bacterial isolates belong to the spore-forming genus Bacillus, although non-spore formers have also been isolated. Theoretically, the smaller a bacterium is, the more likely it is to be carried from Earth to the stratosphere. Ultrasmall bacteria have been frequently isolated from Earth environments, but not yet from the stratosphere. This is an anomalous situation, since we would expect such small bacteria to be over represented in the stratosphere-microflora. Here, we show that ultrasmall bacteria are present in the environment on Earth (i.e., in seawater and rainwater) and discuss the paradox of why they have not been isolated from the stratosphere.
Keywords: aerobiology, filterable bacteria, ultrasmall bacteria, symplasm, panspermia, rainwater bacteria, stratospheric microbes.
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Recent studies have confirmed the original findings of Imshenetsky et al., (1978), that microorganisms (bacteria and fungi) exist in the stratosphere(Wainwright et al., 2003, 2004, Shivaji et al., 2006, Griffin 2008,Yang et al., 2008, Shivaji et al., 2009, Smith et al., 2010). Since a variety of environmental-sampling techniques and approaches to microbiological sample-processing have been used in these various studies we can be confident that the Earth's biosphere does indeed extend into the stratosphere (Wainwright, 2008). While a diverse range of bacteria have been isolated from the stratosphere, spore forming members of the genus Bacillus appear to be particularly common (or else are selectively isolated from this region by the chosen isolation techniques).
Now that that the existence of a stratospheric bacterial has been established the next obvious question is- from where do these organisms originate; from Earth or from space? The application of Occam's razor suggests that since these are microbes are commonly found on Earth they must have an Earth origin. There exists however, the possibility that some, at least, originate from space and that a mixed population of bacteria exists in the stratosphere, some outgoing from Earth and some incoming from space (Wainwright, 2003, Wainwright et al. 2006). How then might the microorganisms, which originate on Earth, reach heights of 60 km above the Earth's surface? One possibility is that they are ejected into the stratosphere by volcanoes. However, at least two of the above cited studies (Wainwright et al., 2003, Shivaji et al., 2006) were conducted some two years after the last major volcanic eruption on Earth; since bacteria and fungi deposit under gravity, any stratosphere isolations of organisms, derived from this study are unlikely to have originated from volcanoes. A number of other mechanisms have been suggested by which bacteria might be carried into the stratosphere, including blue lightening, gravitophotophoresis and electrostatic action (Wainwright et al., 2006, Dehl et al., 2008). However, it appears unlikely that any of these mechanisms would be capable of carrying a particle of a diameter exceeding 1Âµm, i.e. the usual size of bacteria when grown on nutrient-rich laboratory media (Dehl et al., 2008). The likelihood of particles like bacteria being elevated from Earth to the stratosphere is however, likely to increase with decreasing cell size. Very small bacteria do occur in nature (Hahn, 2004) and it is likely that these so-called "ultrabacteria", or "ultrasmall bacteria" (i.e. filterable bacteria) would be more readily easily carried to the stratosphere than bacteria of a larger size. As a result, most of the bacteria which are readily isolated from the stratosphere should be ultrasmall forms. As this is not the case, then either some mechanism must exist by which larger bacteria are carried to the stratosphere or else, the non-ultrasmall bacteria found in this region must have originated from space.
The aim of this study was to determine if ultrasmall bacteria occur in Earth environments, such as soils, seawater and rainwater and attempt to explain the paradox of why they have not been isolated from the stratosphere.
Material and methods
Sampling of soils and seawater
Seawater samples were obtained from the North Sea; the English Channel; the East Pacific Ocean; the Mediterranean Sea; the Atlantic Ocean, the Red Sea and the Black Sea, and the Indian Ocean. The following soil samples were obtained from the UK (Sheffield region): deciduous woodland (under Acer psuedoplatanus and Fagus silvaticus); coniferous woodland soil (Pinus species) agricultural; loam (last crop wheat) and an agricultural grassland soil.
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Fig. 1. The filter apparatus. The seawater sample, or soil suspension, is transferred to the top chamber and is allowed to pass, under gravity, through the micropore membrane between the two chambers into the lower chamber which contain the liquid bacteria-isolation medium.
Seawater samples and soil suspension (1g. soil-100ml of sterile, distilled water) were filtered through a micropore filter apparatus (Fig.1), having either a 0.2 or 0.1 Âµm, micropore filter apparatus(Millipore Express Plus);the apparatus consists of two chambers with a membrane filter between; the seawater sample (100ml) or soil suspension (1g. soil:100ml de sterile, deionized water) was transferred to the top chamber and allowed to pass, under gravity (i.e. without suction), into the lower chamber containing the liquid bacterial isolation medium. The filter apparatus was then incubated at 250C for 2 weeks and checked at intervals for signs of clouding of the medium, i.e. bacterial growth. The following media were used: 1) LB broth; 2) nutrient broth; 3) LB and nutrient broth made up in autoclaved (1200C for 20mins) seawater (for use with seawater samples only) and 4) LB and nutrient broth diluted 100 fold with distilled water (oligotrophic, i.e. low nutrient, media). All media were autoclaved at 1200 C for 20 mins.
Checks for integrity of filter membrane
The integrity of the micropore filter was checked as follows. When a bacterium passed through one of the micropore filter membranes (with the result that the medium belong tuned cloudy) the top part of the filter apparatus was removed and a new one containing fresh medium (Nutrient Broth) was screwed into the upper chamber which contains the filter. A culture of Staphylococcus aureus, grown on Nutrient agar) was then allowed to pass through the filter, under gravity into the medium (Nutrient Broth) below. If no growth of this bacterium occurred (which was always the case), it was taken that the membrane did not allow through "normal" sized bacteria (i.e. exceeding 1Âµm) and that, as a result, only ultrasmall species (i.e., filterable forms) had been isolated.
Selective isolation of Bacillus species
For the selective of members of the genus Bacillus, the seawater and soil suspensions were heated to 800C for 5 mins. a temperature which kills none-spore formers and thereby selectively isolates spore forming bacteria, i.e. mainly Bacillus species (Travers et al., 1987).
Sampling of rainwater
Samples of rain water were collected in open, sterilized containers from the roof of Firth Court, University of Sheffield during December; 2009. Cell masses present in this rainwater were studied using light microscopy as well as environmental scanning electron microscopy (the latter at the University of Manchester). Symplasm was also stained with the DNA stain Sybr Green (which stains fluorescent green in the presence of DNA) and examined using a fluorescent microscope. Rainwater samples were also was passed through 0.2Âµm filters as described above for seawater.
Results and Discussion
Evidence for the occurrence of ultrasmall bacteria in seawater
Bacteria capable of passing through 0.2 Âµm filters were isolated from all of the seawater samples. No such filterable bacteria were however, isolated from the wide range of soil samples tested. These findings agree with previous reports on the widespread occurrence 0.2Âµm- filterable bacteria in seawater from around the world. Other workers have however, reported the isolation of 0.2 Âµm filterable bacteria from freshwaters and soils, a finding which we have been unable to replicate. Only one of these seawater isolates was identified in this laboratory (using 16srRNA analysis); this was a species of Rhienimuria isolated from the North Sea sample.
Attempts to selectively isolate Bacillus spp. and oligotrophic members of this genus
Since Bacillus sp. are the most frequently reported bacteria found in the stratosphere attempts were made to selectively isolate ultrasmall forms of members of this genus from the environmental samples used. It has been reported that the size of laboratory grown bacterial isolates decreases when grown in a nutrient poor (oligotrophic), medium; thus it may be possible to make "normal sized "bacteria filterable merely by growing them in an oligotrophic medium (such as the 100 fold diluted medium used here). No filterable Bacilli were however, isolated from any of the environmental samples used here, and the laboratory strains of Bacillus species (B.sphaericus, B. simplex and B. putilus) were not converted to filterable forms when grown on low-nutrient medium.
Evidence for the presence of ultrasmall bacteria from rainwater
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Ultrasmall bacteria were seen using the light microscope (X100 oil immersion) in samples of Sheffield rainwater occurring in small masses comprising a variety of different morphologies and sizes; these masses are referred here as"symplasm" . This term was used in the early microbiology literature to refer to amorphous mass of bacteria observed in soils; here, the term is re-introduced to refer to cell-masses seen in rainwater. Fig. 2 shows a typical rainwater-derived symplasm mass as seen under the light microscope(X100, oil). A mass of cells of different sizes is seen held together in an amorphous mass-the symplasm.
Fig.2 a) light microscope image of symplasm from rainwater; b) fluorescent (green) microscope image of symplasm as observed after Syber Green II staining.
The obvious question that next arises is- are these symplasm masses biological in nature, or just inorganic particles? To answer this question, the symplasm was stained using Syber Green II stain. This is a fluorescent nucleic acid stain which fluoresces green in the presence of DNA and RNA (Fig. 1b), thereby showing that the particle mass is made of biological entities. This was further confirmed using the environmental scanning electron microscope (at Manchester University) was next used to view the symplasm. The environmental SEM allows biological material to be viewed without pre-coating the sample with metals such as gold.
As can be seen in Fig 3(a) subum particles were seen, some of which are in the region about 0.1 Âµm (note that, compared to the light microscope, a better size determination can be achieved by the use of this microscope).
Fig.3. (a) symplasm viewed under environmental SEM, (b) a hole in one of the symplasm components created by long term exposure to the electron beam of the scanning electron microscope, thereby showing that the structures seen are not inorganic particles.
Next, a technique was developed to demonstrate that the individual symplasm features observed in rain are biological in nature and not merely inorganic particles (i.e., dust). In order to achieved this differentiation, samples of rainwater containing symplasm were exposed to a longer than normal period of exposure of the electron beam. Biological particles, unlike inorganic forms are likely to be affected by such exposure. Figure 3 (b) shows that this is the case, and that the symplasm cells are holed by long-term exposure to the electron beam. This method backs up the Sybr Green II nucleic acid staining approach used here, confirms the fact that symplasm cells are biological. It is also included here in order to highlight a technique which can be used, when studies are restricted to the use of the environmental scanning electron microscope for differentiating between biological and inorganic samples in any environmental sample collected on Earth, or elsewhere.
A wide range of bacteria were isolated from the symplasm masses (using LB and Nutrient media), showing that they are not made up of single species, but are a complex mixture of different bacteria and possibly other microorganisms. No ultrasmall bacteria were however isolated when rainwater containing symplasm masses were passed(without suction) through a 0.2Âµm membranes ,showing that the individual ultrasmall bacterial seen using microscope techniques are firmly bound with in the syplasm masses, which are too large to pass through the filters.
Discussion of these findings in relation to the microbiology of the stratosphere
As was mentioned in the Introduction, a number of recent studies have confirmed that microorganisms, both bacteria and fungi, can be isolated from the stratosphere at heights of up to 60km, with the majority of studies being conducted at 41km. The range of environmental and laboratory-based sampling techniques used in these studies would appear to exclude the possibility that these stratosphere isolates are all contaminants; we can safely conclude therefore that both bacteria and fungi do occur in the stratosphere. Assuming that some, at least, of these stratosphere organisms originate from Earth we need to explain how they can reach these extreme heights. Although the exact mechanisms of such transfer are unknown, it is likely that the smaller the viable microbial unit involved the more chance it would have of being carried to the stratosphere. The fact that ultrasmall bacteria exist on Earth therefore obviously increases the chances of microorganisms being elevated from the surface of the planet to the stratosphere. There is however, one paradox which needs to be addressed in relation to this possibility- namely that the ultrasmall bacteria which have been isolated and characterised belong to the Bacteriodes, Alphaproteobacteria, Betaproteobacteria, Actinobacteria and Spirochaetes (Hahn, 2004), Spirillum, Leucothrix, Flavobacterium, Cytophaga,Vibrio ( Anderson and Heffernan,1965) and, to date, none of these bacteria have been isolated from the stratosphere; the isolated bacterial population of this region being dominated by species of Bacillus. As a result, if the bacterial flora of the stratosphere results from ultrasmall bacteria is transferred from the Earth's oceans to the stratosphere one would expect that the stratosphere microflora would contain, if not be dominated by, species of ultrasmall bacteria which are commonly found on Earth. This finding might suggest that ultra small bacteria are not carried up into the stratosphere and that the bacteria, such as species of Bacillus commonly isolated from this region are in fact incoming from space. Alternatively, the presence of Bacillus species in the stratosphere could follow from a reduction in cell size following starvation in Earth environments which would reduce their size and make them amenable to the mechanisms (known or unknown) which might be capable of transferring bacteria from Earth to heights of 41km and above (our results show however, that this is unlikely in the case of Bacillus species). Differential survival, and or, isolation of Bacillus sp., and the other bacteria found in the stratosphere may offer an alternative explanation. A mixed population of ultrasmall bacteria may be lifted into the stratosphere but only a few (mainly spore-forming Bacilli) might survive in this region, or be readily cultured when stratosphere-derived samples are returned to the laboratory. However, non-spore forming bacteria, such as species of Staphylococcus and Micrococcus and Mycobacterium, have been isolated from the stratosphere (Wainwright et al., 2003, Imshenetsky et al., 1978), so bacteria other than spore formers can clearly survive in this region, as can be seen from the following list of bacteria which have been isolates from the stratosphere to date (Table1).
Table 1. Bacteria isolated from the stratosphere
40km and above
Bacillus isronensis, Bacillus aryabhattai, Bacillus simples Bacillus aerius, Bacillus aerophilus, Bacillus stratosphericus, Bacillus altitudinis,Bacillus pumilus
Micrococccus albus, Mycobacterium luteum, Janibacter hoylei (Actinomycete), Staphylocccus pastueri
Bacillus subtilis, Bacillus endophyticus,
Brevibacterium (Actinomycete) Spp., Microbacteria Spp., Staphylococcus Spp.
None spore forming species are given in bold
We were unable to a) selectively isolate filterable (0.2 Âµm) Bacillus species from any of our environmental samples and b) convert Bacillus species from normal laboratory sized bacteria to filterable bacteria by the imposition of a period of growth on an oligotrophic medium. However, Miteva and Brenchley (2005), reported the isolation (from 120,000 year old Greenland Glacier ice core) of several species of Bacillus isolates(described as being distinctly related to Bacillus mucilaginosus) possessing small filterable cells and spores.
The symplasm masses observed here could have been formed a) on Earth, or at low altitude, or b) at high altitude in the stratosphere. In the first case, the relatively large masses formed are unlikely to be carried into the stratosphere. In the latter case however, individual, ultrasmall bacteria might be more readily transferred from Earth to the stratosphere, where they coalesce to form symplasm masses which then deposit readily to Earth. In this way one can envisage a cycle whereby individual ultrasmall bacteria are carried from Earth into the stratosphere and subsequently deposited to Earth after forming into high density symplasm masses. These masses could then break up (and reproduce) in Earth environments and then be returned to the stratosphere. Such a cycle could provide a mechanism for exposing bacteria to the mutagenic effects of the high levels of UV radiation found in the stratosphere and thereby enhance the rate of bacterial evolution of Earth- bacteria. It could also provide a means by which bacteria are carried across the globe, a likelihood which would be particularly important should any of these symplasm-related ultrasmall bacteria are plant, animal or human pathogens.
The absence in the stratosphere of ultrasmall bacteria commonly found in the oceans of the world suggests that such environments do not provide the main source of bacteria to the stratosphere. It should be borne in mind that Wainwright et al. (2006) provided evidence in support of the view that the stratosphere contains a mixed population of bacteria comprised of those coming in from space (panspermia) and those exiting from Earth (negative panspermia).
The following points summarize our discussion:
By the known mechanisms, it is likely that the smaller the particle is, the more likely it is to be transferred from Erath to the stratosphere.
This suggests that stratospheric microorganisms should be predominantly species of ultrasmall bacteria which occur on Earth.
However, the bacteria which have, to date, been isolated from the stratosphere are members of bacterial genera not typically isolated from Earth environments after filtration, i.e. notably species of Bacillus.
The bacterial growth media used in this study was capable of isolating ultrasmall bacteria from stratospheric samples, if they are present in the stratosphere-the fact that they are not, suggests that stable, ultrasmall bacteria are not present in the stratosphere, even though such small bacteria should be more readily carried from Earth to the stratosphere than are bacteria of size larger than 1micron.
The most likely explanation for the occurrence of non-filterable, "large" bacteria, identical to those found on Earth, is that small, starvation forms are carried, by some mechanism to the stratosphere.
Why representatives of the stable, ultrasmall bacteria, found in the Earth's oceans are not over-represented amongst stratosphere isolates remains an enigma. It could be argued that such bacteria cannot withstand the extreme environmental conditions found in the stratosphere, as can for example resilient, spore-forming Bacilli. However, non-spore forming "large", bacteria have also been isolated from this region. Ultrasmall Bacilli have been found in 1200 year ice cores in Greenland, so ancient ice may provide a source of those Bacilli which have been isolated from the stratosphere.