During several periods in the Earth’s history, extensive sedimentation of halite and some other minerals from hypersaline seas took place. An estimated 1.3 million cubic kilometers of salt were deposited in the late Permian and early Triassic periods alone (ca. 240–280 million years ago;
Zharkov 1981). The continental land masses were concentrated and formed the supercontinent Pangaea. Salt sediments developed in large basins, which were connected to the open oceans by narrow channels. The paleoclimate was warm and arid in a wide belt around the equator, causing large scale evaporation. About 100 million years ago, Pangaea started to break up; the continents were displaced to the North, and folding of new mountain ranges such as the Alps and Carpathians was underway (
Einsele 1992). As a result of these movements driven by plate tectonics, huge salt deposits are found today mainly in the Northern regions of the continents (), e.g. in Siberia, Northern and Central Europe (Zechstein series), South-Eastern Europe (Alps and Carpathian mountains), and the Midcontinent basin in North America (
Zharkov 1981).
The formation of most of the Alpine salt sediments and the Zechstein deposits is dated to the Late Permian period, while some Alpine deposits are dated to the Early Triassic period (see also
Radax et al. 2001 for a detailed description). No significant salt sedimentation had occurred after that period in the pre-Alpidic regions. This is different from some other salt evaporites in Europe; for instance, waters from the receding Tethys sea in the Eastern parts of Eurasia caused salt sedimentation well into the Miocene (about 20 million years ago). The stratigraphic positions of the evaporites, together with the determinations of sulfur isotope ratios, are indicators for the geological age (
Holser and Kaplan 1966). In addition, pollen grains or spores from extinct plants in the sediments, which are often well preserved and exhibit distinct morphological features, have been examined (
Klaus 1974). Both methods confirmed the Permo-Triassic origin of the Alpine and also of the Zechstein salt sediments. The Alpine salt deposits are located today at altitudes between 500 and 1200 m; their thickness is between 200 and 500 m, although some deeply buried layers were estimated to be up to 1000 m. The layers of clay and limestone prevented the washing-out of the salt during the heavy precipitation during the ice ages. Many of the salt deposits in Europe have been mined for centuries, and newly opened mine tunnels and shafts, as well as deep drilling operations provide opportunities for obtaining rock salt samples. shows freshly drilled bore cores, which were used for our investigations.
Dombrowski (1963) and
Reiser and Tasch (1960) were the first to describe viable microorganisms, which were isolated from ancient rock salt, in the 1960s (reviewed by
McGenity et al. 2000). More recently,
Norton et al. (1993) classified isolates from British salt mines of Permian and Triassic age as species of
Haloarcula, Halorubrum, Halobacterium and a variety of new types on the basis of polar lipid composition, and
Gemmell et al. (1998) investigated the evolution of the
Haloarcula representatives by comparing 16S rRNA gene sequences with those of surface isolates.
Vreeland et al. (1998) isolated halophilic bacteria, some of them probably haloarchaea, from the Permian Salado formation in the midcontinent basin in the USA, and from brines close to that formation.
Our group isolated from Permian rock salt, which was collected from the salt mine in Bad Ischl, Austria, numerous colonies with intense pigmentation (), which indicated the presence of carotenoids and bacterioruberin. One isolate was a coccus, growing in clusters, which was designated strain BIp (
Stan-Lotter et al. 1993). Based upon polyphasic taxonomic data, the strain was recognized as a novel species and named
Halococcus salifodinae (
Denner et al. 1994). This was the first isolate from ancient rock salt, which was formally classified and deposited in several international culture collections. Two independently isolated strains, Br3 (from solution-mined brine in Cheshire, England) and BG2/2 (from a bore core from the mine of Berchtesgaden, Germany) resembled
Halococcus salifodinae BIp in many properties; in addition, rock salt samples were obtained eight years later from the same site and several halococci were recovered from these samples, which proved to be identical to strain BIp (
Stan-Lotter et al. 1999). The data suggested that viable haloarchaea, which belong to the same species, occur in geographically separated evaporites of similar geological age. Another halococcal isolate from the Bad Ischl salt formation, which differed from the previously described strains, was subsequently identified as a novel species and named
Halococcus dombrowskii (
Stan-Lotter et al. 2002).
Halococcus salifodinae and
Halococcus dombrowskii have so far not been found in any hypersaline surface waters, or any location other than salt mines. Several non-coccoid strains were later obtained from a freshly drilled bore core at the salt mine in Altaussee, Austria (about 40 km distance from Bad Ischl), which were similar in their 16S rRNA sequence to
Halobacterium salinarum NRC-1; however, other properties were different and consequently, a novel species was created,
Halobacterium noricense (
Gruber et al. 2004). contains a list of the formally classified isolates from alpine rock salt and a strain from a British salt mine. shows the relationships of the haloarchaeal isolates from Permo-Triassic rock salt to several haloarchaeal type species in the form of a phylogenetic tree, based on 16S rRNA gene sequence data.
| Table 2Haloarchaeal isolates from Permo-Triassic rock salt and salt mine brine |
Another example of an isolate from ancient sediments is a single rod-shaped
Halobacterium strain from a 97,000-year-old salt formation in the USA (
Mormile et al. 2003); the isolate was deemed to resemble
Halobacterium salinarum NRC-1. The microbial content of ancient rock salt is generally low - estimates range from 1–2 cells/kg of salt from a British mine (
Norton et al. 1993) to 1.3 × 10
5 colony forming units (CFUs) per kg of alpine rock salt (
Stan-Lotter et al. 2000); nevertheless, the reports showed that viable haloarchaeal isolates were obtained reproducibly by several groups around the world. The data support the hypothesis that the halophilic isolates from subterranean salt deposits may be the remnants of populations which once inhabited ancient hypersaline seas; in addition, they provide strong evidence against the notion that the recovered strains could be the result of laboratory contamination, since the isolates were obtained independently from different locations.
Analysis of dissolved alpine rock salt with molecular methods was also performed by extracting DNA and subsequent amplification and sequencing of 16S rRNA genes. The results provided evidence for the occurrence of numerous haloarchaea, which have not yet been cultured (
Radax et al. 2001;
Fish et al. 2002). Similarities of these 16S rDNA gene sequences were less than 90–95% to known sequences in about 37% of approximately 170 analysed clones (
Radax et al. 2001;
Stan-Lotter et al. 2004); the remaining clone sequences were 98–99% similar to isolates from rock salts of various ages (
McGenity et al. 2000) and to known haloarchaeal genera. These data suggested the presence of a very diverse microbial community in ancient rock salt.
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envelopes and inner structures
