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1. ARTICLE IN PRESS FEMS Microbiology Letters 10778 (2002) 1^5 Microorganisms cultured from stratospheric air samples obtained at 1 41 km 2 a;Ã…
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  • 1. ARTICLE IN PRESS FEMS Microbiology Letters 10778 (2002) 1^5 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⁄ (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.
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