0099-2240/78/0035-001$02.00/0APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 1978, p. 1-5 Vol. 35, No. 1Copyright © 1978 American Society for Microbiology Printed in U.S.A.

Upper Boundary of the BiosphereA. A. IMSHENETSKY,* S. V. LYSENKO, AND G. A. KAZAKOV

Institute ofMicrobiology, USSR Academy of Sciences, Moscow, USSR

Received for publication 13 August 1976

By using meterological rockets fitted with specially designed analyzers, samplesfor microbiological investigation have been taken. The analyzer design preventedextraneous microorganisms from penetrating into the analyzer. Before beingused, the analyzers were sterilized with high gamma-ray doses. For the first timemicroorganisms have been detected in the mesosphere at an altitude of 48 to 77km. The microorganisms are microscopic fungi having black conidia or spores(Circinella muscae, Aspergillus niger, Papulaspora anomala) and one speciesforming green conidia (Penicillium notatum). Colonies ofMycobacterium luteumand Micrococcus albus have also grown. Five of the six species have synthesizedpigments. The presence of pigmented microbial forms leads us to believe thatnatural selection is occurring in the mesosphere because cells possessing chrom-ogenous pigments (carotenoids, melanins) are more resistant to ultraviolet-rayaction. A greater number of microorganisms have been registered in the meso-sphere during dust storms than in the absence of strong winds.

The possibility ofmicroorganisms transfenringand surviving at various altitudes above theEarth's surface has attracted significant interestin view of the development of space biology.This involves the upper boundary of terrestriallife and the problem of planetary quarantine, aswell as the possibility of microbial transport inouter space with cosmic dust and micromete-orites.According to V. I. Vernadsky, the biosphere

implies all the living organisms on the earth intheir relationship with the environment (12). Ashas long been known, microorganisms occupythe extreme boundaries of life because of theirextraordinarily wide distribution in nature andtheir unique resistance to unfavorable physicaland chemical factors. They live in hot springsat 70 to 750C, on the surface of snow in moun-tains, in the soil of deserts, in concentrated saltsolutions, etc. (6). Microorganisms have beenshown to exist in the lower boundaries of thebiosphere as well. A variety of microorganismshas been found in a number of ocean troughsas low as 10 to 11 km. The pink color of watersassociated with the presence of oil, and comingup from great depths, was due to the presenceof purple sulfur bacteria (4, 8).

Solving the question concerning the upperboundary of the biosphere proved to be morecomplicated. Louis Pasteur, the founder of mi-crobiology, was the first to prove, about 100years ago, that there were fewer microorganismsin the air high in the mountains than in the airof Paris streets. Many years later, microorga-

nisms were detected in air samples taken bymeans of airplanes; the altitude at which thesamples were taken was, however, relatively low.A definite step forward was made in the

United States in 1934 when the balloon ExplorerII was launched. A metal cylinder containing asterilized parachute and sampling device wasthrown down from the balloon gondola. In thisway bacterial and microscopic fungi were de-tected as high as 20 km (10). Far less successfulwere the balloon launchings of 1962 and 1964,for before a balloon was filled with helium, itsenvelope had lain on the field ground, and thesoil microorganisms that had been drawn upinto the envelope dropped from it and pene-trated into the samplers, which strongly suckedin the surrounding air (3).That is practically all that has been done in

this field.

MATERIALS AND METHODSSampling for microbiological analysis in the strato-

sphere aid higher is a complicated problem in viewof the methods used. An absolute guarantee shouldbe provided that the samples contain only those mi-croorganisms belonging to the stratosphere or meso-sphere, and any possibility of microorganisms of adifferent origin penetrating the analyzer should becompletely excluded. Because in our case samplinghas been carried out at altitudes of 48 to 85 km,further discussion will deal with the study of themesosphere.

Meteorological rockets reaching as high as 100 kmhave been used as sampling rockets. To exclude apossible bacteriocidal effect of ultraviolet (UV) rays,

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2 IMSHENETSKY, LYSENKO, AND KAZAKOV

the rockets were fired at night. The sampling analyzerwas located in the upper part of the rocket. It wascovered with a removable metal cone at the top. Thecone was heated to 500 to 600'C when the rocket wasrising, which resulted in all the microorganisms beingkilled on both the inner and outer cone surfaces. Assoon as the rocket reached the altitude of sampling,the cone detached itself as a result of pyrocartridgeexplosion. Figure 1 depicts the instrument bay, withthe analyzer and the detachable cone. The removalof the cone was followed by the removal, by means ofpyrocartridge explosion, of the analyzer metal coverthat had hermetically sealed it. Then the analyzerseparated from the rocket and continued ascending.

For this analyzer a long polyethylene-teriphtolatefilm 80 mm in width was manufactured. Its surface,except for the edges, was covered with viscous nutrientmedium consisting of equal proportions of meat-pep-tone broth and wort, to which 15% polyvinyl alcoholwas added to make the medium viscous. The film wasthen covered with a similar film having no nutrientmedium on it, and the two films were hermeticallyglued together at the edges. The films were kept inan autoclave at 0.5 atm for 30 min. The glued filmswere wound around a rotating roll. For sampling, the

FIG. 1. Instrument bay with an analyzer (left) anda rocket cone (right) detachable in flight. Polyethyl-ene bags holding the analyzer can be seen on top ofthe analyzer.

upper film was separated from the lower one and waswound around a roll located above it. The lower film,covered with the viscous nutrient medium, was pulledwith a speed of 4.3 to 4.5 m/min. In all, 1.3 to 3.2 mof film was pulled, and both mineral particles andmicroorganisms precipitated on the viscous nutrientmedium. Then another sterile polyethylene-teriphto-late film free of nutrient medium was unwound froman upper rotating roll, and the edges of the two filmswere hermetically glued together and wound arounda rotating roll. Later the instrument package wasejected and descended by parachute. The ascendingvelocity of the rocket and the analyzer ranged from500 to 1,000 m/s, the descending velocity of the ana-lyzer being 6.2 m/s. Sampling by means of the analyzerwas carried out during the ascent stage of the rockettrajectory only.The films with nutrient medium 'were mounted in

the analyzer, which was enclosed in two polyethylenebags and was sterilized by gamma irradiation for 9 h(3.2 to 3.5 Mrads at 98.9 rads/s).The analyzer was fitted with a programming device,

allowing all the necessary operations to be carried outin a certain sequence. After the analyzer landed, thehermetically sealed films were transported to the lab-oratory and put into a thermostat. Systematic obser-vations of the transparent films with a low-magnifi-cation microscope made it possible to detect growingcolonies of microorganisms.

RESULTSExperiments. (i) Rocket launching. Sam-

pling in the mesosphere, using meteorologicalrockets and analyzers, took place in desert re-gions of the Kazakhstan Republic, which haspoor plant cover, the vegetation period beingover by June. The soil is dustlike, with occa-sional stony patches. Constant winds in the re-gion blow at 4 to 7 m/s. Sometimes the velocitysharply rises as high as 20 m or more per s,bringing about dust storms that result in theraising of both soil particles and soil microorga-nisms.The first run of experiments was conducted

in May and June 1974, immediately after a duststorm that lasted for 7 h. The second run ofexperiments was carried out in October and No-vember 1974. In both cases three rockets bearinganalyzers were launched. Samples were takenat different altitudes ranging from 48 to 85 km.The flight periods of the analyzers from the timeof the rocket launching to that of the landingof the analyzer did not exceed 1 h.The rocket flights were radar tracked during

all the flight trajectory. The landing coordinatesof the instrument bay with the analyzer werereported to the search crew, who reached thelanding place by car or helicopter (Fig. 2). Theinstrument bay was transported to the labora-tory and dismantled there. The glued fllms werewithdrawn and placed into a sterile, almost her-

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metically sealed cassette.(ii) Number of microbial colonies grown.

The films withdrawn from the analyzers wereput into the sterile cassettes, transported to thelaboratory, and kept in a thermostat at 28 to300C for 14 days. In the mesosphere, bacterialcells, conidia, and fungal spores, and possiblyeven fragments of fungal mycelium, had pene-trated the transparent nutrient medium betweenthe glued films. They began to germinate, andcolonies resulted. The glued films were exam-ined by a projection microscope speciallyadapted for these particular studies, and, be-cause it was possible to pul the nutrient mediumfilms, one could observe magnified microbialmicrocolonies on the screen. After two such ob-servations of the films, the grown colonies wereexamined with an optical microscope. Such col-onies were photographed before the films wereseparated. Figure 3 shows a fungal colony thatgrew between the two films on the transparentnutrient medium. Colonies developed in the

middle part of the films, and no germinationwas seen on the edges.

In two of the six rockets launched the ana-lyzers failed to operate after discarding thecones. Table 1 summarizes the results obtainedby means of rocket-borne analyzers. No coloniesgrew on the nutrient medium film in rocket 3.This, in particular, confirmed the sterilizationprocess and inability of microorganisms to pen-etrate from the rocket instrument bay holdingthe analyzer into the lower nutrient mediumfilm. Thirty-one microbial colonies grew on thenutrient medium films. The miniimum altitudeat which microorganisms have been detected is48 km; the maximum is 77 km. Microscopicobservation of the colonies revealed both fungiand bacteria growing on the hermetically sealedlower nutrient film.The analyzer in rocket 1, which was launched

immediately after a dust storm, detected a sig-nificantly larger number of microorganisms thanthose in rockets 2, 3, and 4, which were fired in

FIG. 2. An instrument bay landing.

FIG. 3. Growth of individual colonies between glued films.

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TABLE 1. Results with rocket-borne analyzers

Rocket no. Sampling altitude(kmn)1 48-582 61-703 61-754 57-77

77-85

No. of coloniesgrown

203080

autumn when no dust storms took place.Characteristics of the microbial cultures

detected in the mesosphere. The films con-taining visible colonies were treated (wiped)with 15% hydrogen peroxide on both sides andthen transferred into a sterile box, where pieceswith colonies were cut out of the film and putinto sterile petri dishes 18 to 20 cm in diameter.The zone of peripheral colony growth was 50 to60 mm from the cut. Then the upper and lowerfilms were separated with sterile instruments.The grown colonies were inoculated on meat-

peptone agar and wort agar slants in test tubes.In all cases, the colonies that grew between thefilms contained mycelium of one species only,which was evident from microscopic observationof the material taken from the colony after in-oculation and from numerous inoculations ondense nutrient media. The tubes were then keptin a thermostat at 280C for 7 to 14 days.

In all the cultures obtained, the morphology,development history, and physiology were stud-ied, and all the data necessary for the determi-nation of their taxonomy were collected. On thewhole, four microscopic fungi, one bacterial spe-cies, and one mycobacterial species were iso-lated. The isolated cultures are commonlyknown species, i.e., the bacteria Micrococcusalbus and Mycobacterium luteum and the fungiCircinella muscae, Aspergillus niger, Penicil-lium notatum, and Papulaspora anomala.The most important conditions for obtaining

microbial cultures are the following: thoroughsterilization of the analyzer, faultless operationof the film-pulling device, and precipitation ofparticles from the mesosphere onto the filmcovered with viscous nutrient medium. Thatonly microorganisms from the mesosphere pre-cipitated on the film is proved by the followingfacts. Sporiferous bacteria usually detectedwhen using imperfect sterilization techniqueswere absent. Also, colonial growth was only inthe middle part of the glued films and not attheir edges. Three of the four species of grownmicroscopic fungi developed black spores; onespecies formed green spores. Of the bacterialspecies, one also had pigmentation. Imshenetskyhas shown (5) that pigmented microbial formstend to predominate in the air, and this corre-

sponds to their higher UV radiation resistance.Colorless forms (leucoraces) from pigmentedcultures isolated from air were obtained experi-mentally, and they lost their relatively high UVradiation resistance (5). The resistance role ofblack pigmentation is known to be higher thanthat of various carotenoids. The colonies of twospecies of microscopic fungi were black, and onespecies had a grey colony; their melanins pro-tected them from a UV radiation effect. UVradiation intensity is much higher in the meso-sphere than in the air near the Earth's surface.Natural selection of pigmented forms of micro-organisms quite likely takes place in the meso-sphere. Pigment-free cells perish more readily,which explains the predominance of pigmentedforms in the mesosphere. From the availabledata, the weight of microbial cells may be con-sidered to be smaller than that of dust particlesdetected at an altitude of 100 km. Noctilucentclouds found at a height of 60 to 80 km areknown to contain mineral particles of both ter-restrial and cosmic origin. Figure 4 demonstratesthat mineral particles have been detected withinthe altitude range of 75 to 100 km (4). By com-paring the densities and dimensions of the min-eral particles and microorganisms detected, onecan determine the latter to be lighter (Fig. 4).

DISCUSSIONFor many years, scientists have considered

the "import" of life to the Earth from otherplanets or space a possibility. The possibility ofmicrobial transport in space itself is not objec-tionable; the theory of panspermia, however, isnot scientifically grounded because it does notsolve the problem of the origin of life. The latest

H.-

FIG. 4. Detection of microorganisms and mineralparticles in the mesosphere. The shaded parts of thecolumns and solid black lines correspond to the al-titude at which the presence ofmineralparticles andmicroorganisms has been established.

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developments in the fields of cosmochemistryand organic synthesis have greatly contributedto the theory of the origin of life. The presenceof microbes in meteorites has been experimen-tally examined, and it has been shown that ter-restrial microorganisms penetrate into mete-orites soon after the fall of the latter (7). Hence,"comers" from space are unlikely to be foundin meteorites that have fallen to Earth. Micro-bial transport through space is possible and hasbeen proven by the presence of viable microbeson the Moon, which were transferred by spacevehicles that had landed there earlier (11). Thetransfer of microorganisms in space has beendifficult to explain. The cells of commonlyknown species are larger and heavier than theparticles capable of transferring them in spaceunder the action of light pressure. At present,in connection with the discovery of a numberof microorganisms invisible through an opticalmicroscope, it has become obvious that the tin-iest microbes and viruses can be transferred invacuum under light action since being insidespace dust particles fully protects them fromthe bactericidal effect of UV radiation.The detection of the six microbial species in

the mesosphere makes it important to study itshigher layers further as well as to sample in theionosphere. Air currents are capable of many

things. Let us recall, for instance, that CharlesDarwin observed insects fall on the deck of theBeagle when it was rather far from Britain (2).

LITERATURE CITED

1. Aksenov, S. I. 1972. Problems of space biology, p. 7, vol.19. M. Nauka Publishers, Moscow.

2. Darwin, C. R. 1935. Naturalist voyage around the world.(In Russian) M.-L. Gosizdat. Biol. Med. Lit. 1:139.

3. Greene, V. W., P. D. Pederson, D. A. Lundgren, andC. A. Hagberg. 1964. Proceedings of the AtmosphericBiology Conference, p. 199.

4. Hemenway, C. L, and R. K. Soberman. 1962. Astron.J. 67:256.

5. Imshenetaky, A. A. 1946. Mikrobiologiya 15:422.6. Imahenetaky, A. A. 1975. Principles of space biology

and medicine, p. 271, vol. 1. M. Nauka Publishers,Moscow.

7. Imshenetsky, A. A., and S. S. Abyzov. 1970. Extrater-restrial life and its detection, p. 157. M. Nauka Publish-ers, Moscow.

8. Isachenko, B. L. 1951. Selected papers. M.-L. Issled.Akad. Nauk SSSR 2:213.

9. Malyshek, V. T., and A. A. Maliyantz. 1935. Dokl.Akad. Nauk SSSR 3:221.

10. Rogers, L. A., and F. C. Meier. 1936. U.S. Army AirCorps stratosphere flight of 1935 in the balloon "Ex-plorer II," p. 146. The National Geographic Society,Washington, D.C.

11. Silverman, G., E. F. Munoz, and V. I. Oyama. 1971.Nature (London) 230:19,169.

12. Vernadsky, V. L. 1926. Biosphere. L. Nauchn. Khim.Tekhn. Issled.

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