Isolation and Identification

For isolation of photoautotrophic microorganisms, prolonged enrichments (8-18 weeks) of thawed but otherwise undisturbed permafrost samples under continuous illumination (1,000 lx) were applied. The enrichment cultures in BG11 (Rippka 1988), Bristol (Gollerbakh and Shtina 1969), BBM (Brown and Bold 1964) media were incubated at 4 and 20°C. Enrichments were re-examined weekly to document biodiversity (Table 6.1).

Isolates were initially examined by measuring of the fluorescence excitation spectra at 686 nm (Vishnivetskaya et al. 2001). Identification of algae and cyanobacteria was based on morphological (Komarenko and Vasil'eva 1978; Rippka et al. 1979; Andreeva 1998) and phylogenetic (Nubel et al. 1997; Krienitz et al. 2003) criteria. DNA was extracted from cyanobacteria (Smoker and Barnum 1988) and green algae (Fawley and Fawley 2004). Bacteria-specific (8F and 1492R) (Weisburg et al. 1991) and cyanobacteria-specific (CYA106F and CYA781R) (Nubel et al. 1997) primers were used to amplify 16S rRNA gene from cyanobacteria. The 18S rRNA gene from the green algae was amplified with primers NS1 and 18L (Gilichinsky et al. 2007b).

Table 6.1 The observation frequency of viable bacteria, cyanobacteria and green algae within Siberian permafrost

Observation frequency3 (%)

Table 6.1 The observation frequency of viable bacteria, cyanobacteria and green algae within Siberian permafrost

Observation frequency3 (%)

Sediment

Age (years)

Bacteria

Cyanobacteria

Green algae

Lake-swamp loam

Holocene (1,000-10,000)

91

17

50

Alluvium sandy

Late Pleistocene

80

9

18

loam

(20,000-30,000)

Channel-fill sands

Late Pleistocene

40

0

0

(20,000-30,000)

Marine (littoral)

Middle Pleistocene

40

0

0

sands

(100,000-200,000)

Lake-alluvium

Middle Pleistocene

90

8

39

loam and sandy

(200,000-600,000)

loam

Lake-alluvium

Late Pliocene-early

38

6

15

loam and sandy

Pleistocene (0.6-1.8

loam

millions)

Lake-alluvium

Late Pliocene-early

44

13

9

loam and sandy

Pleistocene

loam

(2-3 millions)

aTwo hundred and ninety three Siberian permafrost samples were studied; the observation frequency is expressed as a percentage of samples with viable microorganisms aTwo hundred and ninety three Siberian permafrost samples were studied; the observation frequency is expressed as a percentage of samples with viable microorganisms

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