Lysak L.V.*, Soina V.S., Kudinova A.G *, Lapygina E.V., Petrova M.A.**

* Lomonosov Moscow State University, Soil Science Faculty

** Institute of Molecular Genetics RAS

Detailed soil investigations in Antarctica are concentrated mainly on soil genesis, development of relatively precise soil maps and molecular-

genetic analysis of microbial components in such soils. Molecular-genetic methods showed the diversity of bacteria in Antarctic soils, but these

results don’t reveal biological activity, physiological status and stability of microbes in external environment. Detailed soil investigations in

Antarctica are concentrated mainly on soil genesis, development of relatively precise soil maps and molecular-genetic analysis of microbial

components in such soils. Molecular-genetic methods showed the diversity of bacteria in Antarctic soils, but these results don’t reveal biological

activity, physiological status and stability of microbes in external environment. A combination of culture-depended and independent techniques

was used to characterize bacteria community in earlier not investigated soils in East Antarctic Coast. The samples of soils and barren rock with

endolithic soil-like bodies were taken from the inter-hill wet valleys area (oasisis) in East Antarctica). The bottoms of inter-hill valleys show

maximum biota concentration and highest bio- and soil diversity. Moss, lichen and algae ground covers are formed here, as well as algal-bacterial

mats and microorganisms develop various soil profiles in sandy granitoid sediments. The main feature of these soils is that they are formed under

the protection of a gravel pavement, or detritus. Under such "armor» the effect of wind is decreased, the moisture is better preserved, as well as

the proportion of direct light with aggressive UV component is reduced. Microbiological studies revealed the following features of examined

Antarctic soils:

:

Soil at the wet valley of

Larsemann Hills oasis beyond

the meltwater flow.

Source of moisture – snow

patches.

(Site М3): GP- gravel

pavement,

GPalgae – green algae and

cyanobacteria in the sandy

bedding of gravel pavements

1. The total number of bacterial cells counted by staining with acridine orange was rather high for Antarctic

habitat and varied in the range of more than 10 mln cells/g.

Total number of bacteria, AO staining, sites 10-45, M3, M2 Total number of bacteria, AO staining, sites 10-15L1, 10-15P1, 10-06

2. Using Live/Dead (L7012) pigment stain it was shown that 60% of detected total number cells (in some samples

about 80%) have undamaged cell membranes that testify for their viability and stability to extreme environment.

Share of potentially viable cells, sites 10-45, M3, M2 Share of potentially viable cells, sites 10-15L1, 10-15P1, 10-06

4. Considerable number among viable cells (70-80%) were nanoforms with cell diameter not higher than 200 nm, that

exceed such indexes in temperate soils and represent the specific pool of Antarctic bacteria.

Nanoforms of Antarctic bacteria as viewed by transmission electron microscopy

Quantitative results

Weig

ht%

0

10

20

30

40

50

C O Na Mg Al Si S Ca

Biofilms in Antarctic samples

Larsemann Hills, E. Antarctica

Quantitative results

Weig

ht%

0

10

20

30

40

50

C O Na Mg Si S Ca

Share of “nanoform” cells with diameter of no more than 200 nm, AO

staining, sites 10-45, M3, M2

8. The alternation of 10 to 30 cycles of freeze-thawing of all soil samples resulted

in an increase in the number of cultured heterotrophic bacteria (CFU), as

compared with the number of CFU, counted immediately after thawing, in the

most cases, one order of magnitude higher. Several freeze-thawing cycles can

be used for more complete extraction of cultivated forms from Antarctic soils. It

is possible, that increase in CFU indexes occurs due to partial destruction of

biofilms.

7. Taxonomic composition at the domain level and phylum determined by

molecular biological method revealed representatives of the phylum

Proteobacteria, Actinobacteria, Planctomycetes, Acidobacteria. Domain

Archaea was presented in smaller quantities than Bacteria, its proportion was

less than 20%, as compared with the domain Bacteria accounted for about 80%

of all cells identified by FISH. Application of FISH method (fluorescence in situ

by hybridization) allowed to determine the representatives of different genera -

the same among bacteria with common size cells, and nanoforms, that testify

for the bacterial transformation to nanoforms under unfavourable conditions.

6. The concentration of bacterial biomass in studied soils was significantly (ten

or more) lower, than that in the soils of temperate zone, and varied depending

on the type and extent of vegetation on the surface of the soil and the depth of

sampling. The maximum concentration values of microbial biomass were

observed in organic horizons (6.4-13.5 mg/g oven-dry soil), the lowest in the

mineral horizons (1.1-2.30 mg/g). Stocks of bacterial biomass calculated for the

entire soil profile ranged from 0.13 mg/m2 in endolithic soil to 5.67 mg/m2 in the

soil with abundant growth of hypolithic algae and cyanobacteria.

9. Examined soils were characterized by significant discrepancy between indexes

of total and viable number of cells, as well as high irregularity in horizons of

developing soils. It can be explained both by specification of physical and

chemical processes in permanently frozen habitats and by imperfection of

microbial isolation techniques. The basic form of the existence of cryptogamic

organisms in investigated soils – are biofilms, which can be detected at the micro

and submicro levels and possess an extracellular polymer matrix. It is a natural

form of the development of organisms in subaerial conditions on mineral

surfaces especially in extreme conditions. Such data indicate the importance of

taking into account in Antarctic investigations the microbial biofilms, which may

play the leading role both in soil development and modification of the cell

metabolism that require special techniques for reactivation of bacteria from such

extreme environment.

10. Antibiotic resistance in microbes is widespread and it has been demonstrated

earlier that soil bacteria especially in contaminated soils are rich in resistance

determinants to both natural and synthetic antibiotics commonly used in clinics.

Systematic studies of antibiotic resistance among bacteria isolated from

uncontaminated soils have not yet fully been explored. Antarctic soils are

considered as the most nonpolluting environment with minimal anthropogenic

action. The first experiments were carried out to compare resistance spectrum of

bacteria isolated from Antarctic and contaminated soils. It was shown that 70% of

Antarctic bacteria revealed high resistance both to natural and synthetic

antibiotics, especially they demonstrated multiple resistance to various

combinations of antibiotics. Some of these combinations were unique for

particular sites of Antarctic soils. For the first time it was shown that antibiotic

multiple resistance does not be attributed only for contaminated soils. Antibiotic

resistance in Antarctic soils can be explained by innate resistance formed in

extreme Antarctic climate conditions. Collection of isolated Antarctic bacteria can

be used to reveal evolution processes in mechanism of antibiotic resistance in

nature.

3. The largest number and proportion of viable cells was observed in the fine earth directly under the gravel pavement.

Such habitats (sand beddings with moss pads, algae and micromycetes colonies) are the most favourable for the

development of bacteria, as sheltered by the gravel pavement from wind corrasion, dehydration and aggressive UV

radiation, but at the same time they are close to the surface, well warmed by pavement insolation and are fed by the

melting snowfields.

Share of “nanoform” cells with diameter of no more than 200 nm, AO

staining, sites 10-15L1, 10-15P1, 10-06

5. The total number of bacteria does not find a strong correlation with a carbon content (correlation coefficient 0.15),

while the number of heterotrophic bacteria by the number of CFU, vice versa, show such connection (correlation

coefficient 0.78). This indicates that a significant portion of the organic matter is slightly binded to the mineral matrix

and primarily contained in labile form, available for heterotrophic bacteria. Scale -200nm

ABUNDENT AND DIVERSITY OF PROKARIOTIC COMMUNITIES

IN SOILS OF OASISES IN EAST ANTARCTICA.