Development of Isolation Technique

The majority of known techniques for microbial isolation below the freezing point are based on using liquid media with glycerol or other antifreeze compounds (Breezee et al. 2004). The lowest temperature limit for isolates obtained by this approach was -10 to -12°C. The disadvantage of using supercooled liquids is obvious. First, it is technically unreliable at temperatures below -7°C, some flasks turn frozen for seemingly unknown reasons. Secondly, we cannot proceed to the lower and extremely challenging temperatures which are expected to dominate in the polar desert or outside the Earth. Thirdly, homogeneous liquid media are fine for aquatic bacteria, but often are inappropriate for terrestrial habitats such as soils and permafrost. We developed a solid-state cultivation system (Panikov and Sizova 2007) which can be used at any below-zero temperature, and more closely imitates natural growth conditions in permafrost. Solid-state cultures are grown as thin frozen film between plastic sheets or in powder of microcrystalline cellulose with ethanol or other appropriate C-sources, volatile or soluble. We never detected significant subzero degradation of cellulose or other polysaccharides. Probably, the degradation of polymeric compounds requiring the synthesis of extracellular hydrolytic enzymes is completely arrested in frozen media.

Conventional liquid and new solid-state enrichments led to the isolation of different organisms: liquid media resulted in the isolation of bacteria similar to those described in studies of polar aquatic habitats, while solid frozen media allowed the isolation of yeasts and mycelial fungi (Table 9.2). Apart from ethanol, aerobic growth in frozen media was supported by H2 and succinate. The isolated bacteria belong to new species, but are closely related (95-99% of similarity in 16S or 26S rDNA genes) to known psychrophilic bacteria and fungi recently isolated from sea ice and Antarctic habitats (Polaromonas, Arthrobacter, Mrakia, Cryobacterium). The most interesting bacteria Polaromonas hydrogenovorans is able to grow autotroph-ically on the mixture of H2 and CO2 or heterotrophically on succinate, pyruvate, and citrate.

Surprisingly, the most active growth in frozen media was displayed by eukaryo-tic microorganisms, dimorphic yeasts of the genus Leucosporidium and ascomycet-ous fungi of the genus Geomyces. The last organisms grew exponentially at -8°C,

Table 9.2 Psychrophilic and psychrotolerant microorganisms isolated from Alaskan permafrost and top soil

Organism, strain

Isolation source

Enrichment conditions

Growth temperature (°C) Min Max

Closely related phylotypes (BLAST)

Genbank accession number

Pseudomonas sp. 3-2005

Forest soil, Fairbanks, 10-20cm, frozen 9 months/year

Liquid ethanol -mineral medium, 0°C

-15

25

Antarctic bacterium R-9113 isolated from lake mat (96%)

DQ 094182

Arthrobacter sp. 9-2

Permafrost, Fairbanks, 50-55 cm

Liquid ethanol-mineral medium, 0°C

-15

25

Arthrobacter sp. Anl6 isolated from deep sea sediment (98%)

DQ 094184

Polaromonas hydrogenovorans

Forest soil, Fairbanks, 10-20cm, frozen 9 months/year

Liquid mineral medium, H,:CO, in headspace, 0°C

-1

25

Polaromonas naphtalenevorans (99%)

DQ 094183

Leucosporidiales spp. MS-l.MS-3

Forest soil, Fairbanks, 10-20cm, frozen 9 months/year

Ethanol-MCCa solid media frozen to -5°C and -8°C

-18

20

Cryptococcus sp. Ytty94 Y24 (99%), Leucosporidium scottii isolate (97%)

DQ 295018

Mrakia sp. MS-2

Forest soil, Fairbanks, 10-20cm, frozen 9 months/year

Ethanol-MCC solid media frozen to -5°C

-16

18

Mrakia sp. and M.frigida, isolated from various Antarctic habitats (100%)

DQ 295019

Geomyces spp. FMCC-1, FMCC-2, FMCC-3, FMCC-4

The same

The same, -8°C

-35

18

Geomyces pannorum from cryopegs (98%); Aleurodiscus farlowii Burt, wooddecomposing fungi (100%)

DQ499471 - 74 (ITS region) DQ520619-22 (LSU rRNA)

*MCC microcrystalline cellulose

*MCC microcrystalline cellulose

Fig. 9.10 Demonstration of competitive advantage of fungi over bacteria while growing on solidstate frozen media (after (Panikov and Sizova 2007) ). Left: Growth dynamics of Arthrobacter sp. 9-2. 50 |il of bacterial suspension were frozen in plastic bags 2 x 6" (=inches) with ethanol-mineral medium (EMM), rolled into a tube and placed in a Hungate tube. Growth was followed from the rate of CO2 production. The legend indicates the growth temperature. Right: Growth dynamics of an eukaryotic consortium in frozen ethanol-MCC powder. The eukaryotic consortium (Geomyces spp. - Leucosporidium spp.) was used as inoculum of 29 tubes containing EMM with cellulose powder. Growth was followed at -8°C as CO2 production rate. Note that six out of 29 tubes displayed higher growth rates than other slow growers. Heavy solid curves are the best-fit exponential equation which ignores auto-oscillations

Fig. 9.10 Demonstration of competitive advantage of fungi over bacteria while growing on solidstate frozen media (after (Panikov and Sizova 2007) ). Left: Growth dynamics of Arthrobacter sp. 9-2. 50 |il of bacterial suspension were frozen in plastic bags 2 x 6" (=inches) with ethanol-mineral medium (EMM), rolled into a tube and placed in a Hungate tube. Growth was followed from the rate of CO2 production. The legend indicates the growth temperature. Right: Growth dynamics of an eukaryotic consortium in frozen ethanol-MCC powder. The eukaryotic consortium (Geomyces spp. - Leucosporidium spp.) was used as inoculum of 29 tubes containing EMM with cellulose powder. Growth was followed at -8°C as CO2 production rate. Note that six out of 29 tubes displayed higher growth rates than other slow growers. Heavy solid curves are the best-fit exponential equation which ignores auto-oscillations with a generation time of about 1 week; under further cooling the growth rate and respiratory activity progressively declined, but were still detectable at the lowest tested temperature of -24°C. For comparison, prokaryotic organisms (Pseudomonas sp and Arthrobacter sp.) grew in solid media only in a progressively declining fashion (not exponentially), indicating the presence of some unknown restriction factor (Fig. 9.10).

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