1. ARTICLE IN PRESS
FEMS Microbiology Letters 10778 (2002) 1^5
www.fems-microbiology.org
Microorganisms cultured from stratospheric air samples obtained at
1
41 km
2
a;Ã
, N.C. Wickramasinghe b , J.V. Narlikar c , P. Rajaratnam d
M. Wainwright
3
F
4 a
Department of Molecular Biology and Biotechnology, University of She⁄eld, She⁄eld S10 2TN, UK
O
b
5 Cardi¡ Centre for Astrobiology, Cardi¡ University, 2 North Road, Cardi¡ CT10 3DY, UK
c
Inter-University Centre for Astronomy and Astrophysics, Post Bag 4, Ganeshkhind, Pune 411 007, India
6
d
O
Indian Space Research Organisation, Antariksh Bhavan, New Bel Road, Bangalore 560 094, India
7
8 Received 24 September 2002; received in revised form 31 October 2002; accepted 5 November 2002
PR
9 First published online
10 Abstract
11 Samples of air removed from the stratosphere, at an altitude of 41 km, were previously found to contain viable, but non-cultureable
D
12 bacteria (cocci and rods). Here, we describe experiments aimed at growing these, together with any other organisms, present in these
13 samples. Two bacteria (Bacillus simplex and Staphylococcus pasteuri) and a single fungus, Engyodontium album (Limber) de Hoog were
TE
14 isolated from the samples. Although the possibility of contamination can never be ruled out when space-derived samples are studied on
15 earth, we are confident that the organisms originated from the stratosphere. Possible mechanisms by which these organisms could have
16 attained such a height are discussed.
17 ß 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Microbiological Societies.
EC
18
19 Keywords : Exobiology ; Astrobiology ; Panspermia; Extreme environment
20
R
21 In an earlier paper Harris et al. [6] reported the discov- 40
1. Introduction
ery of clumps of cocci-shaped sub-micron-sized particles 41
R
22 The extension of the biosphere upwards to include var- of overall average radius 3.0 Wm from isolates of ¢lters 42
23 ious levels of the atmosphere has been discussed intermit- that were recovered from an earlier stratospheric probe. 43
O
24 tently for many years, particularly in relation to the trans- The clumps were identi¢ed, as bacteria, ¢rst using a scan- 44
25 port of pathogenic microorganisms from one part of the ning electron microscope and later using an epi£uores- 45
C
26 globe to another [1]. The occurrence of microorganisms in cence microscope. The latter technique used a mem- 46
27 cumulous clouds is not in dispute, nor is their role in brane-potential-sensitive dye (carbocyanine) and 47
N
28 nucleating atmospheric ice crystals [2,3] as such organisms £uorescence was interpreted as revealing the presence of 48
29 are likely to be of terrestrial origin. viable cells. Here we describe studies aimed at isolating 49
U
30 Attempts were made to probe the stratosphere in the and growing these previously viable, but non-cultureable 50
31 years immediately prior to the space age [4]. Although it bacteria. 51
32 was claimed that bacteria and fungi can be found over the
33 altitude range 18^39 km, such results were generally dis-
34 missed on the basis of contamination. 52
2. Materials and methods
35 Narlikar et al. [5] sought to repeat these early experi-
36 ments using rigorous sterilisation protocols, combined 2.1. Stratosphere sampling 53
37 with state-of-the-art balloon experimentation technology
38 used in India for research in atmospheric physics as well Air samples were collected over Hyderabad, India on 20 54
39 as cosmic ray and infrared astronomy. January 2001 at various heights up to 41 km. The collec- 55
tion involved the deployment of balloon-borne cryosam- 56
plers of the type described by Lal et al. [7]. The cryosam- 57
1
pler comprised of a 16-probe manifold, each probe made 58
2 * Corresponding author. Tel. : +44 (114) 222 4410.
3 of high-quality stainless steel capable of withstanding pres- 59
E-mail address : m.wainwright@she⁄eld.ac.uk (M. Wainwright).
1 0378-1097 / 02 / $22.00 ß 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Microbiological Societies.
2 PII: S 0 3 7 8 - 1 0 9 7 ( 0 2 ) 0 1 1 3 8 - 2

2. ARTICLE IN PRESS
2 M. Wainwright et al. / FEMS Microbiology Letters 10778 (2002) 1^5
60 sures in the range 1036 mb (ultravacuum) to 200 b. The (PDA) and Czapek Dox. Nutrient-free silica gel was also 114
61 probes and all their components were thoroughly steri- included in an attempt to isolate oligotrophic microorgan- 115
62 lised. They were £ushed with acetone and heat sterilised isms [8]. The extracts were spread over the surface of the 116
63 to temperatures of 180‡C for several hours. The entrance medium by gentle hand motion (not by the use of spread- 117
64 to each probe was ¢tted with a metallic, motor driven er). The plates were then incubated at 25‡C and examined 118
65 (Nupro) valve that could be opened and shut on ground periodically. Microscope studies involved the use of an 119
66 telecommand. The payload trailed at a shallow angle of optical microscope (U1000, oil emersion, phase contrast) 120
67 elevation behind the balloon gondola, being tethered by a and by scanning electron microscopy (SEM) after gold 121
68 sterilised 100-m-long rope. As a further precaution against coating. 122
69 the possibility of collecting any traces of out-gassed mate-
70 rial from the balloon surface, a sterilised intake tube 2 m 2.3. SEM 123
71 long formed an integral part of the cryosampler ensemble.
F
72 The intake to each probe was covered during the balloons Sterile, distilled water was added to the surface of fresh, 124
O
73 ascent through the atmosphere. inoculated PDA plates, which were gently swirled gently 125
74 Throughout the £ight the probes remained immersed in by hand. Samples of the water extract were then asepti- 126
O
75 liquid Ne so as to create a cryopump e¡ect, allowing am- cally removed using a sterile syringe, such that the Petri 127
76 bient air to be admitted when the valves were opened. Air dish lid remained as close to the base as possible. The 128
77 was collected into a sequence of probes during ascent, the extract was transferred to a sterile membrane ¢lter appa- 129
PR
78 highest altitude reached being 41 km. The cryosampler ratus (Nalgene, 100 ml, 0.2 Wm) and ¢ltered using low 130
79 manifold, once the probes were ¢lled, was parachuted suction. The membranes were then removed and trans- 131
80 back to ground. The probes were stored at 380‡C until ferred to a sterile Petri dish and allowed to air dry. They 132
81 the laboratory work began. were then examined using SEM. 133
D
82 Laboratory analysis relating to only two probes is dis-
83 cussed here: 2.4. Precautions taken against contamination 134
TE
84 1. Probe A: Collection between 30 and 39 km altitude, a
85 total quantity amounting to 38.4 l of air at NTP. Standard microbial techniques, employing a laminar air 135
86 2. Probe B: Collection between 40 and 41 km altitude, a cabinet, were used throughout. All plates were used within 136
87 total quantity amounting to 18.5 l of air at NTP. 8 h of pouring and were checked for contamination, both 137
EC
88 Two procedures were used to extract aerosols aseptically by light microscope examination and by culturing. Control 138
89 from the probes: membranes were also analysed after being subjected to 139
90 1. Procedure 1: The air from the exit valve of each probe identical transfer procedures used for stratosphere-derived 140
91 was passed in a sterile system in a micro£ow cabinet samples. In addition, the isolation experiments were re- 141
R
92 sequentially through a 0.45-Wm and a 0.22-Wm micro- peated in a separate laboratory, by a technician who was 142
93 pore cellulose nitrate ¢lter (¢lter diameter 47 mm). not informed of any expected outcomes. 143
R
94 2. Procedure 2: Following the completion of Procedure 1,
95 the probes were injected with sterile phosphate bu¡er
O
96 solution, agitated for several hours in a shaker to dis- 144
3. Results and discussion
97 lodge particles adhered to the walls, and the liquid sy-
C
98 ringed out and passed sequentially through three ¢lters: After 4 days incubation no bacterial colonies were seen 145
99 (i) 0.7-Wm glass micro¢bre ¢lter, (ii) 0.45-Wm cellulose on any of the inoculated plates independent of the medium 146
N
100 acetate ¢lter, and (iii) 0.2-Wm cellulose acetate ¢lter. used (including nutrient-free silica gel). In order to deter- 147
101 Most of the aerosols are expected to have been collected mine if bacteria were present, but not forming colonies, 148
U
102 in Procedure 1. the surface of the media was gently removed using a sterile 149
scalpel and viewed under the optical microscope. Bacteria 150
103 2.2. Microbial isolation studies (coccus and rod forms) were seen only in surface medium 151
removed from the PDA plates. The coccus was seen more 152
104 Membranes, stored at 380‡C were aseptically trans- frequently than the rod and occurred singly or in clumps 153
105 ferred (using a laminar air low cabinet) to sterile, plastic of two or three cells. The rod was not seen when in the 154
106 universal bottles, and left at ambient (15^20‡C) temper- samples of PDA examined under the SEM. The coccus in 155
107 ature for 4 days. Sterile, distilled water (10 ml) was added contrast was seen under the SEM occurring in clumps of 156
108 to each tube and allowed to soak the membranes for 1 h. coccoid cells (0.5^2.0 Wm diameter, Fig. 1). It is notewor- 157
109 The tubes were then shaken vigorously on an orbital shak- thy that the viable, but non cultureable, organisms seen 158
110 er for 5 min. Samples (0.5 ml) of the water extracts were (using SEM) by Harris, et al. [6] consisted of clumps of 159
111 then transferred to the surface of the following media cocci, and an occasional rod. 160
112 (Oxoid, autoclaved at 120‡C): Columbia base; Mueller Samples of surface medium obtained from undisturbed 161
113 Hinton; L Broth; nutrient agar; potato dextrose agar plates were then removed and transferred to L-broth (in- 162

3. ARTICLE IN PRESS
M. Wainwright et al. / FEMS Microbiology Letters 10778 (2002) 1^5 3
F
O
O
1 Fig. 1. SEM photograph of coccoid cells removed from surface of PDA. Bar represents 1 Wm.
PR
163 cubated with shaking, at 30‡C for 4 days). Liquid medium involved in the isolation of these bacteria since the only 201
164 was removed from any tubes showing turbidity and plated samples of this medium available to us at the time was 202
165 (in the standard manner) onto LB medium that was then approximately 15 years old, and showed signs of browning 203
166 incubated at 30‡C until bacterial growth appeared. After due to oxidation. When prepared, the powder produced a 204
D
167 transfer to LB medium (incubated for 2 days at 30‡C)
168 bacterial colonies appeared which comprised rod and a
TE
169 coccus. The rod was large and formed long chains and,
170 after extended incubation, formed spores. The coccus or-
171 ganism occurred singly, in pairs or clusters. Both organ-
172 isms were Gram-positive, non-acid fast, and catalase-pos-
EC
173 itive, and facultatively anaerobic. The coccus was initially
174 distinguished from Staphylococcus by its ability to grow
175 on furazolidone (Sigma, 100 Wg ml31 ), tentatively indicat-
176 ing it to be a species of Micrococcus ; the rod was tenta-
R
177 tively identi¢ed as a species of Bacillus. The cultures were
178 independently identi¢ed using 16S rRNA analysis (by
R
179 NICMB, Aberdeen, using the MicroSeq1 database). The
180 coccus and rod were identi¢ed respectively as Staphylococ-
O
181 cus pasteuri (99.9% similarity) and Bacillus simplex (100%
182 similarity). The B. simplex isolate is phylogenetically
C
183 closely related to Brevibacterium frigoritolerans (Fig. 2a);
184 while the S. pasteuri isolate, while being closely related to
N
185 Staphylococcus warneri, is relatively phylogenetically dis-
186 tinct from Staphylococcus epidermidis (Fig. 2b). Further
U
187 details of the characteristics of B. simplex and S. pasteuri
188 are respectively given in Priest et al.[9] and Chesneau et
189 al.[10].
190 No organisms were isolated using nutrient-free silica gel
191 medium. This suggests that oligotrophs were absent or, if
192 present, were incapable of growing under the physical^
193 chemical conditions provided by the medium.
194 In addition to the two bacteria, a single fungus was
195 isolated on the PDA plates. It was identi¢ed by CABI
196 Bioscience (Egham) as Engyodontium album (Limber) de
197 Hoog [11]. This fungus appeared on a single isolation
198 plate; no other fungi were isolated on any other media.
199 Bacteria were only observed growing on, and isolated 1
Fig. 2. Dendrograms showing phylogenetic relations of (a) the rod-
200 from, the surface of PDA. An element of serendipity was 2
shaped isolate (NCSQ 16861 and (b) the coccus (NCSQ 11681).

4. ARTICLE IN PRESS
4 M. Wainwright et al. / FEMS Microbiology Letters 10778 (2002) 1^5
3.2. Discussion of a possible Earth origin for the isolates 259
205 brown medium that set, but which was softer than both
206 normal PDA, and the other media employed. It is possible
It is generally accepted that few particles of Earth origin 260
207 that the oxidised, soft characteristics of this medium may
can cross the tropopause, a natural barrier occurring at 261
208 have encouraged bacterial growth; it is noteworthy that
around 17 km above the Earth’s surface. While convection 262
209 soft (semi-solid) agars have been used before in bacteriol-
currents mix ground level particulates in the air and read- 263
210 ogy, to isolate organisms [12].
ily carry them into the troposphere, temperature inver- 264
sions beyond 15 km lead to barriers through which very 265
211 3.1. Comments on the origin of the isolated organisms
few aerosols can penetrate. Whenever rare events such as 266
volcanic eruptions loft particles above 30 km, particles 267
212 Since these organisms are found on earth it is impossible
larger than a few microns fall back quickly to the ground 268
213 to state categorically that they are not contaminants.
under gravity. The isothermal temperature regime between 269
214 However, every e¡ort was made, within the limits of our
F
15 and 25 km e¡ectively stops the ascent of particulates, 270
215 resources, to avoid contamination and to check at every
O
and the rapidly rising ambient temperature gradient at 271
216 stage that all materials were sterile and free from contam-
higher levels should make the upper stratosphere almost 272
217 ination. For example, none of the organisms were isolated
O
impervious to the transport of aerosols from the ground. 273
218 on non-inoculated plates left exposed to the atmosphere in
A volcanic origin for the bacteria sampled in this study 274
219 the laminar air-£ow cabinet, or from breath plates and
is ruled out for the simple reason that there was no vol- 275
220 glove or skin washings. Similarly, no contaminants were
PR
canic eruption recorded in a 2-year run-up to the balloon 276
221 found on any of the media, sterile water, or control mem-
launch date on 20 January 2001, and for reasons already 277
222 branes used in this study. A technician (who was unaware
stated, a settling rate at 0.18 cm s31 from 41 km, as calcu- 278
223 of any expected outcome and working in a separate labo-
224 ratory) also repeated the isolation protocol and isolated lated by Colbeck [16] would drain out particles of 3 Wm 279
D
225 the same two bacteria seen by us. Finally, the isolates are radius in a matter of weeks. A similar objection applies to 280
226 not common laboratory contaminants and have never rare meteorological events. Assuming our collections on 281
TE
227 been used in any of the laboratories involved in these 20 January 2001 gave us representative stratospheric sam- 282
228 studies. It is particularly noteworthy that none of the bac- ples at 41 km no process that is purely terrestrial can 283
229 terial isolates formed colonies on any of the initial media sustain the high densities of bacterial clusters as are im- 284
230 employed, as would have been the case with air contam- plied, such densities would require an in-fall, or fall-back 285
EC
231 inants. The balance of probabilities would therefore sug- rate of a factor of 1 t year31 over the entire planet [6]. 286
232 gest that the organisms were indeed obtained from the
233 stratosphere. 3.3. Discussion of a space origin for the isolates 287
234 The survival of microorganisms in the stratosphere will
R
235 be particularly limited by exposure to UV light. It is gen- An extraterrestrial origin of the isolates [17] provides a 288
236 erally accepted that spore-forming bacilli, such as B. sim- consistent, if controversial, explanation of our ¢ndings. 289
R
237 plex are relatively resistant to such radiation, as are bac- The bacterial material, cultured in the present experiment, 290
238 teria whose vegetative cells tend to clump together; and detected earlier through £uorescence microscopy, can 291
O
239 essentially because the outer cells provide a UV barrier be regarded as forming part of the 100 t day31 input of 292
240 that protects the inner cells. Whisler [13] found that Sar- cometary material known to reach the Earth. Critics of 293
C
241 cina lutea, which forms UV protective packets of cells, is panspermia may argue that 3 Wm radius particles get burnt 294
242 100 times more resistant to UV than is Escherichia coli, through frictional heating and end up as meteors. Some 295
N
243 while B. subtilis and Staphylococcus aureus are respectively fractions may do, but others would not. Survival depends 296
244 three and eight times more resistant. It is not surprising of many factors such as angle of entry and mode of de- 297
U
245 therefore that our stratospheric bacterial isolates exhibit position in the very high stratosphere. Several modes of 298
246 potentially UV-resistant morphologies. The environment entry can be considered that permit intact injection into 299
247 found at 41 km will obviously be extreme, not only in the stratosphere, possibly starting o¡ as larger aggregates 300
248 terms of UV exposure, but also because of low temper- released from comets that disintegrate into a cascade of 301
249 atures and pressures. At ¢rst sight, it would appear un- slow-moving smaller clumps at heights above 270 km 302
250 likely that microorganisms could withstand such extremes. where frictional heating would be negligible. Evidence 303
251 However, Streptococcus mitus has been shown to survive for such disintegrations has been available for many years 304
252 for 31 days on the Moon’s surface, while Bacillus subtilis [18], and more recent studies of particles collected using 305
253 has been recovered in a viable state after 6 years of ex- U2 aircraft have also shown the survivability of extremely 306
254 posure to the space environment [14,15]. fragile organic structures. 307
255 If, as seems likely, the microorganisms isolated here Many microbiologists will probably react negatively, as 308
256 were obtained from the stratosphere, how did they get we initially did, to the suggestion that the any stratospher- 309
257 to a height of 41 km? The two obvious sources are (a) ic bacterial £ora would include species of the genus Staph- 310
258 from Earth and (b) from space. ylococcus. This view is prejudiced by the preconception 311

5. ARTICLE IN PRESS
M. Wainwright et al. / FEMS Microbiology Letters 10778 (2002) 1^5 5
312 that Staphylococci are solely human pathogens; in reality 356
References
313 however, members of this genus are hardy organisms that
357
[1] McCarthy, M. (2001) Dust clouds implicated in the spread of infec-
314 can exist in a variety of natural environments.
358
tion. Lancet 358, 478.
315 The main theoretical limitation on the view that micro-
359
[2] Jayaweera, K. and Flanagan, P. (1982) Investigations on biogenic ice
316 organisms, such as B. simplex, S. pasteuri and E. albus, 360
nuclei in the arctic atmosphere. Geophys. Res. Lett. 9, 94^97.
317 which are found on Earth, arrive from space does not 361
[3] Bigg, E.K. (1983) Particles in the upper atmosphere. In: Fundamen-
318 however, relate to problems concerned with survival. In- 362
tal Studies and the Future of Science. (Wickramasinghe, C., Ed.), pp.
363
38^51. University College Cardi¡ Press, Cardi¡.
319 stead, such a bias relates to fundamental genetic and evo-
364
[4] Burch, C.W. (1967) Microbes in the upper atmosphere and beyond.
320 lutionary considerations. This is because it is considered
365
Symp. Soc. Gen. Microbiol. 17, 345^373.
321 di⁄cult to reconcile prevailing ideas on the evolution of 366
[5] Narlikar, J.V., Ramadurai, S., Bhargava, Pushpa, Damle, S.V.,
322 microorganisms and recognised phylogenetic relationships 367
Wickramasinghe, N.C., Lloyd, D., Hoyle, F. and Wallis, D.H.
323 with the view that organisms, identical to those found on 368
F
(1998) The search for living cells in stratospheric samples. Proc.
369
SPIE Conf. 3441, 301^305.
324 Earth, are continually entering the system from another
370
[6] Harris, M.J., Wickramasinghe, N.C., Lloyd, D., Narlikar, J.V., Ra-
O
325 source; except that is if evolution occurs elsewhere at rates
371
jaratnam, P., Turner, M.P., Al-Mufti, S., Wallis, M.K., Ramadurai,
326 and in the direction which are identical to evolution on 372
S. and Hoyle, F. (2001) The detection of living cells in stratospheric
O
327 Earth, or if terrestrial evolution is driven from outside. It 373
samples. Proc. SPIE. Conf. 4495 (in press).
328 is noteworthy however, that bacteria with genome sequen- 374
[7] Lal, S., Archarya, Y.B., Patra, P.K., Rajaratnam, P., Subbarya, B.H.
375
and Venkataramani, S. (1996) Balloon-borne cryogenic air sampler
329 ces essentially identical to those of modern bacteria have
PR
376
experiment for the study of atmospheric trace gases. Ind. J. Radio
330 been isolated in salt crystals of known ancient provenance
377
Space Phys. 25, 1^7.
331 [19,20]. Such studies have created considerable controversy 378
[8] Parkinsion, S.M., Wainwright, M. and Killham, K. (1989) Observa-
332 since they, like our ¢ndings, would suggest that either the 379
tions on the oligotrophic growth of fungi on silica gel. Mycol. Res.
333 isolates were contaminants or that evolutionary and phy- 380
93, 529^534.
381
D
[9] Priest, F.G., Goodfellow, M. and Todd, C. (1988) A numerical clas-
334 logenetic microbiology is more convoluted than is gener-
382
si¢cation of the genus Bacillus. J. Gen. Microbiol. 134, 1847^1882.
335 ally thought [21].
383
[10] Chesneau, O., Morvan, A., Grimont, F., Labischinski, H. and El
TE
384
Solh, N. (1993) Staphylococus pasteuri sp. nov., isolated from human,
385
animal and food specimens. Int. J. Syst. Bacteriol. 43, 237^244.
336 386
4. Conclusions [11] de Hoog, G.S. (1978) Notes on some fungicolous hyphomycetes and
387
their relatives. Persoonia 10, 33^81.
EC
388
[12] Zobell, C.E. and Meyer, K.F. (1937) Growth zones of Brucella in
337 Studies such as these, in which space-derived samples
389
semi solid medium. J. Bacteriol. 35, 112.
338 are returned to earth for microbial sampling can readily 390
[13] Whisler, B.A. (1940) The e⁄cacy of ultra-violet light sources in kill-
339 be dismissed on the basis that any organism isolated must 391
ing bacteria suspended in air. Iowa State Coll. J. Sci. 14, 215^231.
340 be Earth-based contaminants, even though the balance of 392
[14] Darling, D. (2001) Life Everywhere. Basic Books, New York.
R
393
[15] Hornek, G. (1998) Exobiological experiments in Earth orbit. Adv.
341 evidence suggests that this is not the case in our study.
394
Space Res. 22, 317^326.
342 Future planned cryoprobes will hopefully help to demon-
395
R
[16] Colbeck, I. (1998) In: Physical and Chemical Properties of Aerosols
343 strate that our results are repeatable. However, the only 396
(Colbeck, I., Ed.). Blackie, London.
344 certain means of proving the existence of microorganisms 397
[17] Hoyle, F. and Wickramasinghe, N.C. (2000) Astronomical Origins of
O
345 in the stratospheres is to send probes where samples can 398
Life: Steps towards Panspermia. Kluwer, Amsterdam.
399
[18] Clemett, S.J., Maechling, C.R., Zare, R.N., Swan, P.D. and Walker,
346 be analysed in situ in space; hopefully the present studies
400
C
R.M. (1993) Identi¢cation of complex aromatic molecules in individ-
347 will encourage the sending of such probes in the not too
401
ual interplanetary dust particles. Science 263, 721^725.
348 distant future. 402
[19] Vreeland, R.H., Rosenzweig, W.D. and Powers, W.D. (2000) Isola-
N
403
tion of a 250 million year old halotolerant bacterium from a primary
404
salt crystal. Nature 407, 897^900.
405
U
[20] Fish, S.A., Shepherd, T.J., McGenity, T.J. and Grant, W.D. (2001)
349 Acknowledgements
406
Recovery of 16s ribososmal RNA gene fragments from ancient halite.
407
Nature 417, 432^436.
350 We are indebted to Professor K. Kasturirangan, Chair- 408
[21] Maher, B. (2002) Uprooting the tree of life. Scientist 16, 27^28.
351 man ISRO, for his encouragement and his unstinting sup- 409
352 port of this work. We are also grateful to Professor Ian
353 Colbeck for discussions, Melanie Harris, for help in sam-
354 ple preparation and David Trott for independently repli-
355 cating some of the experiments.