FEMS Microbiology Letters 116 (1994) 189-194
© 1994 Federation of European Microbiological Societies 0378-1097/94/$07.00
Published by Elsevier
189
FEMSLE 05835
Microorganisms surviving for 5300 years
K. H a s e l w a n d t e r * and M.R. E b n e r
Department of Microbiology, University of Innsbruck, Technikerstrasse 25, A-6020 Innsbruck, Austria
(Received 8 November 1993; accepted 14 December 1993)
Abstract: Recently, the well-preserved corpse of a prehistoric man with an age of approximately 5300 years bp was discovered in
the Central European Alps. Analysis of materials associated with the individual has revealed the presence of microorganisms which
are believed to have survived since the time of death. So far, two fungi have been isolated and identified as species of the genera
Chaetomium and Absidia, respectively. In addition, we have obtained one bacterial isolate which we have identified as a
Streptomyces species. Our findings demonstrate that microorganisms can remain viable under appropriate circumstances for
thousands of years. The isolates may enable us to study evolutionary trends within microorganisms.
Key words: Longevity; Absidia; Chaetomium; Streptomyces; Survival
Introduction
On September 19th, 1991, the frozen body of a
prehistoric man from the late neolithic was discovered on a glacier of the Central Alps in Europe (46°46'N 10°51'E) [1,2]. Together with the
body of the 'ice-man', a number of other items of
organic material were encountered. Judged from
the quality and the condition of preservation of
the corpse and the artefacts, they must have
remained frozen over the entire period of time
[3]. The finds associated with the corpse included
a grass mat and leather boots padded with hay.
Radiocarbon dating of grass samples has revealed
an age of approximately 5300 years [4]. Since
dried grass normally carries a specific microflora
* Corresponding author. Tel.: (0512) 218 5180; FAX: (0512)
218 5188.
SSDI 0 3 7 8 - 1 0 9 7 ( 9 3 ) E 0 5 2 7 - J
[5], it was appropriate to investigate whether microorganisms have survived in these materials for
about 5300 years. In order to minimize the risk of
isolating microorganisms which did not originate
from the authentic microflora, the experimental
approach involved isolation of specific microorganisms from the grass samples.
Materials and Methods
The 'ice-man' and all the accessories were
found at 3210 m above sea-level, and were transported to the University of Innsbruck within a
few days of their discovery where they were stored
at 0-4°C. The finds included leather boots padded
with hay which were transported to Innsbruck as
intact entities. From the central and previously
unexposed part of the padding material, subsampies were taken on October 2nd, 1991, under
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sterile conditions in a dissecting room where normally autopsies are being carried out. The grass
samples were either washed in 1/4 strength sterile Ringer solution or directly transferred onto
agar plates. The following nutrient media were
used: nutrient agar (= NA, Difco 0001-01), Czapek-Dox agar (= CD, BBL 11140), malt extract
agar (= ME, Merck 5391) and skim milk agar
(= SM, Oxoid L31). The plates were incubated at
18, 45, 50 or 60°C, respectively. The temperature
range between 45 and 60°C was included to allow
for growth of therm0philic microorganisms normally associated with hay [5]. Standard methods
were employed for identification of the various
isolates [6,7].
Results and Discussion
Table 1 lists the microbial isolates obtained,
and their origin. Two of the three isolates are
thermophilic microorganisms. Because it is very
unlikely that all the material has been contaminated with thermophilic microorganisms during
excavation and transport, this fact suggests that
the isolates are the original occupants of the
substrate, which is approximately 5300 years old.
Further support for this assumption is provided
by the fact that such microorganisms are normally
absent from the air spora at high elevation. Despite bi-weekly sampling for 2 years at a nearby
site at 1960 m (distance 16.5 km, 46°52'N ll°2'E)
Absidia and Chaetomiurn spores were never encountered [8]. The same study revealed in high
and especially low elevation alpine sampling sites
a relatively high frequency of spores of Cladosporium, Penicillium and other fungal genera all of
which are known to be potential colonizers of
plant material. If the excavated plant material
had been contaminated, it is most likely that it
would have been by these fungi which are found
in the atmosphere more frequently than Absidia
and Chaetomium. Absidia corymbifera (Cohn)
Sacc. & Trotter (Fig. 1) occurs worldwide in a
range of habitats, and has been isolated from a
variety of substrates including hay [6]. This species
has been described as being both thermophilic
and psychrotolerant [9]. Such features may explain its ability to survive in the ice of a glacier
for about 5300 years.
Chaetomium globosurn Kunze ex Steud., in
contrast, is mesophilic rather than thermophilic
with a temperature range for ascoma formation
and mycelial growth between 18 and 30°C [10].
This species is known to be involved in the decomposition of plant material [6].
Fig. 2 demonstrates the ascoma and spore
formation by this fungus on the grass. The high
number of ascomata formed by this fungus over
the whole length of the blade indicates a high
colonization density. This, again, is a strong indication of its autochthonous and original presence
in the substrate, and that its occurrence on the
blade is not the result of contamination. Interest-
Table 1
Microbial isolates obtained from 5300-year old dried grass
Microorganism
Substrate
Isolation procedure
origin
Isolation method
Growing conditions
Dried grass/
right boot
Direct plating
Czapek-Dox agar,
45°C
Dried grass/
left boot
Direct plating
Malt extract
agar, 18°C
Dried grass/
left boot
Ringer solution
suspension
Skim milk agar,
nutrient agar, 45°C
Zygomycete
Absidia corymbifera
Ascomycete
Chaetomium
globosurn
Actinomycete
Streptomyces
violaceoruber
2-5 cm long blades were either directly transferred to nutrient medium, or washed in 5 ml 1/4 strength Ringer solution of which,
subsequently, 0.1 ml was plated.
191
Fig. 1. Absidia corymbifera colonizing at 5300-year-old grass blade (a) and producing sporangiophores with typical columella and
funnel-shaped collar (see arrow in b; bar = 10/zm). Incubation: 3 days at 45°C on Czapek-Doxagar.
ingly, C. globosum has been reported to survive in
the form of ascospores for more than 10 years in
a dried condition [11], and in high-alpine soil with
long snow coverage [12].
Based on a number of features, some of which
are shown in Fig. 3, the bacterial isolate was
identified as the actinomycete Streptomyces uiolaceoruber (Waksman and Curtis 1916) Pridham
1970 [7]. The isolated strain fails to grow at 60°C,
hence
being thermotolerant rather than truly
thermophilic [13]. Streptomyces species are known
to be frequently associated with dried grass, their
spores becoming abundant in hay [14]. Streptomycetes can exist in soil for long periods as
resting arthrospores [15], hence, their survival in
association with grass under ice for about 5300
years is conceivable.
Under specific circumstances, plant seeds, bacteria and fungi remain viable over a considerable
period of time [16-20]. Samples from lake sediments deposited approx. 1965-2765 years ago
contained viable Thermoactinomyces endospores
Fig. 2. Chaetomium globosum forming ascomata with coiled terminal hairs (a) and ascospores (b) on a grass blade after 3 - 5 weeks
of incubation at 18°C on malt extract agar. Bar in a represents 1 mm.
192
Fig. 3. Streptomycesviolaceoruber isolated from grass blade rinsed with 1/4 strength Ringer solution, 0.1 ml of which was plated on
nutrient agar. Incubation: 1 week at 45°C. (a), morphologyof the aerial mycelium, spore chains forming spirals. (b), scanning
electron micrographof arthrospores. Bar = 1/zm.
[21]. T. sacchari could be isolated from a 9000year-old sediment, a feature which renders this
species as probably one of the oldest known living
organisms [22]. With an age of approximately
5300 years, the isolates obtained in this study are
somewhat younger. They are, nevertheless, still of
interest, especially as our knowledge of the
longevity of fungal spores is scanty. As the organisms isolated in this study have survived for approximately 5300 years, they represent the oldest
living fungi known so far.
Our finding that in addition to Absidia and
Chaetomium, Streptomyces also remained viable
for about 5300 years is also worth stressing, especially as even younger isolates of other Actinomycetes such as T. vulgaris and T. dichotomica
have been found to differ from those regarded as
typical of currently isolated strains [21]. In addition to the vertical transmission of genomes from
one generation to the other, the acquisition of
D N A from other organisms leading to a horizontal spread of genetic information seem to be
important factors in microbial evolution [23]. Microorganisms, in general, have shorter generation
times than higher organisms. This increases the
likelihood that the effects of vertical and horizontal genome transmission become obvious. Therefore, isolates of ancient microorganisms such as
those obtained in this study may prove useful for
investigating evolutionary trends, especially as this
has been claimed for isolates of Therrnoactinomyces which were less than 2000 years old [19].
Acknowledgements
We thank Professor D.J. Read, FRS, for his
comments on the manuscript, and Professor J.
Klima for the scanning electron micrograph.
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