Biological researches

POLYRESISTANCE and SUPERRESISTANCE
of ANTARCTIC MICROORGANISMS
to HIGH CONCENTRATIONS of HEAVY METALS

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Presentation (pdf)

Ukrainian microbiological researches in Antarctic Region are carried out at financial and logistical support of the National Antarctic Scientific Centre of Ukraine. Slide 1.

Microbiological researches in Antarctic Region are included into Ukrainian State scientific program of complex Research of Antarctic biota – i.e. both marine, and terrestrial biocenoses. Slide 2.

In 2001 we have begun complex researches of Antarctic microbial cenoses on biogeographical polygon on island Galindez. Biogeographical polygon is an Antarctic oasis characterized with vertical thermal stability because the highest point makes 58 m above the sea level. Slide 3.

All typical terrestrial Antarctic biotopes are represented at the polygon – mosses, lichens, a grass, micro-pounds, biofilms, etc. Slide 4 (Video: Island Galindez: Biogeographical Research Polygon).

Native and the frozen biological samples were packed into sterile packages and then investigated in Kiev at the Institute of Microbiology and Virology of the National Academy of Sciences of Ukraine. Slide 5.

We selected samples mainly on territories with minimal anthropogenous influence. Most samples were selected in inaccessible areas of polygon in extreme conditions. Slide 6 (Video: Extreme investigations on the Polygon).

3-D model and the topographical map of polygon were developed by means of GIS-methods. Position of sampling points were determined with the help of GPS. Slide 7.

First we did not plan special research of Antarctic metal resistant microorganisms. For studying of Antarctic microbial cenoses we used classical methods of soil microbiology, – the qualitative and quantitative characteristic of microorganisms of a cycles carbon, nitrogen, sulfur, etc. 

At first we have applied heavy metals only as the stressful factor to the quantitative characteristic of a homeostasis of microbial populations. 

We supposed, that the toxic effect of metals will be shown at their concentration from 1 to 10 ppm. It is well-known for soil and water microorganisms. Slide 8.

To our great surprise appeared, that soil microorganisms have shown high resistance to toxic heavy metals. So, concentration of alive microorganisms made about 1 million cells in 1 g of soil even at 1000 ppm of rather toxic soluble form of Cu²⁺ ions. Slide 9.

Moreover, number of viable cells was decreased insignificantly at increase of copper concentration from 200 up to 1000 ppm.

Whereas Cu²⁺ is one of the most toxic metals, we used it as representative, model metal for studying the phenomenon of metal resistance. Slide 10.

Copper resistant microorganisms were discovered in all dominating biotopes of polygon, – on rocks of a glacier, rocky cliffs, in soils, mosses, lichens, lake sediments, etc. Slide 11.

Moreover, microorganisms of majority samples of polygon were resistant to 100 ppm of Cu²⁺. They are pointed with red color on the map of the polygon. Slide 12.

This correlation is well represented by means of factor of distribution of samples containing Cu²⁺ resistant microorganisms. The increase of concentration of toxic metal is usual results in decreasing of number of metal – resistant microorganisms (the left figure).  

For microorganisms of biogeographical polygon the opposite correlation is observed (the right figure).  

This relation is well-defined with the help of Kd-coefficient of dispersion of samples with Cu²⁺ resistant microorganisms. Slide 13.

After we have found out very unusual phenomenon of metal resistance of microorganisms on polygon, we have expanded a research area and a spectrum of toxic metals.

Resistance of the Antarctic microorganisms to 6 most toxic metals – bivalent cations of Hg²⁺, Cd²+, Сu²⁺, Co²⁺, Ni²⁺, Cr(VI) in form CrO₄^2- in a range obviously bactericidal concentration from 50 up to 5000 ppm is investigated. 

Area of researches – is an internal island shelf of passage Penola, 60 kms from the North to the South and 30 kms from the West to the East. Samples are selected on 19 islands and capes of continent. During last 3 years 462 native and frozen samples are selected.  

In all investigated objects presence of microorganisms growing at bactericidal concentrations of toxic metals is revealed.

Concentrations of heavy metals the determined in soils, humus, lake sediments with atomic-adsorption method, were rather low. Therefore the phenomenon of metal resistance is impossible to explain by presence of high metal concentration in an environment. Slide 14.

High level of resistance to increasing concentration of toxic metals is typical for the majority of objects and biotopes.

In most cases toxic metals in the range from 10 to 50 ppm lead to reduce number of cells on solid nutrient medium on 1 or 2 orders, from (ten in the seventh degree) 10⁷ up to (ten in the fifth degree) 10⁵ CFU.

However the further increase of metal concentration in a nutrient medium up to 500 ppm did not reduce number alive cells.  

For mercury maximum permissible concentration made from 50 to 100 ppm. Slide 15.

It is well known, that bacteriostatic or bactericidal concentrations of cadmium, copper, chromium, etc. make from one tenth (0,1) to 10 ppm for heterotrophic microorganisms isolated from the environment.

So, the data received on superersistance of the Antarctic microorganisms to heavy metals do not correspond to the standard point of view about toxicity of metals.

Therefore with the purpose of definition of the maximum permissible concentration of toxic metals for microorganisms we have applied method of thermodynamic prognosis of interaction of microorganisms with toxic metals that we developed in 1988.

The method is based on several simple positions:

1. The microbial metabolism is possible only in a zone of thermodynamic stability of water, in a range of standard redox-potential from - 414 up to +814 mV. (A)

2. All kinds of interaction of microorganisms with metals are possible only in a zone of thermodynamic stability of wate – (B)

3. Toxicity of metals - oxidizers (Hg²⁺, Сu²⁺ and CrO₄^2-) is determined by value of their standard redox-potential.
   Mercury is the most toxic, because it has the highest redox-potential - +920 mV.
   Toxicity also is proportional to concentration of metal in a solution. (С1 – С3)

4. However increasing of metal concentration in 8 orders (from 10 nanomoles (1×10^-8) up to 1,0 М) leads to insignificant increase of redox-potential - only on 200-400 mV, which value is in a zone of thermodynamic stability of water (С1 – С3).

5. From here follows, that for Hg²⁺, concentration of ten milimoles (1×10^-2 M) i.e. 10000 ppm is critical, and for CrO₄^2- and Сu²⁺ growth of microorganisms is theoretically allowable at 1,0 M concentration of these metals. Slide 16.

Inspired with such optimistic forecast, we isolated from the Antarctic soil a number collection of strains, resistant to a wide spectrum of metals in very high concentration.

It is current opinion, that the Antarctic Region is the most non-polluted region on the Earth. Therefore completely mysterious was an existence of microorganisms, resistant to ultrahigh concentration of the most toxic metals in Antarctic Region. Slide 17. 

Despite of very high resistance to chromium(VI), increase of its concentration from 500 to 40000 ppm caused full suppression of growth of microorganisms. 

However adding glucose (20 g/l) into nutrient medium as an additional source of carbon and energy allowed 2 strains grow even at more than 1.0 M i.e. at 60000 ppm chromium concentration. Slide 18.

At present we have isolated more than 50 Antarctic bacterial strains resistant to very high concentration of toxic metals. 10 of them have shown the maximal resistance to three most toxic metals. Slide 19.

The phenomenon of polyresistance i.e. ability to grow at presence in medium of several toxic metals in high, "bactericidal" concentration is typical for all of 10 strains. For example, 3 strains are able to grow on medium containing simultaneous 500 ppm Сu²⁺ 100 ppm of Cr(VI) and Hg²⁺. Slide 20.

High number of metal resistant microorganisms in typical biotopes – soils, mosses, lichens, lake sediments, and humus is very important evidence of their wide occurrence in Antarctic environment. For example, microorganisms, resistant to 500 ppm Cd²⁺ and Cr (VI), are found out in the range from 40 to70 % of biotopes. Slide 21.

For studying occurrence of metal resistant microorganisms of 12 objects we used such 2 quantity indicators (indexes) as:

 – N_CFU – number of metal resistant microorganisms in 1 g of sample,
and C_OC – coefficient of occurrence of metal resistant microorganisms – i.e. ratio of metal- resistant microorganisms to total number of microorganisms - in %. Slide 22.

These indexes allow characterizing quantitative parameters of metal resistance in a complex. Percent of metal resistant microorganisms C_OC gives representation about their relative occurrence, and N_CFU – about their absolute number in biotopes and samples.

Coefficient C_OC is represented in brown color, and value N_CFU – as colored columns.

So, despite of low average values of C_OC from one tenth (0,1) up to 3-5 %, the absolute number NCFU of metal
resistant microorganisms is rather high and equal ten in the third degree (n×10³) or ten in the sixth degree (n×10⁶).

In all 17 investigated objects, in each gram of a sample there are thousands and millions cells of the microorganisms, able to grow at "superbactericidal" concentrations of 6 most toxic metals. Therefore it is possible to suppose, that metal resistance is the widespread phenomenon in the investigated part of Antarctic Region. Slide 23.

The Antarctic microorganisms not only grow at ultrahigh concentration of 6 most toxic metals, but also interact with them. The main ways of interaction is a reduction of metals up to insoluble forms, forming insoluble compounds with microbial metabolites, and also mobilization of insoluble forms of metals. Slide 24.

The received results allow drawing the following conclusions.

1. For the first time it is shown, that resistance of microorganisms to ultrahigh concentration of toxic metals is widely occurred phenomenon in area of an internal island shelf of the Western Antarctic Region.

2. Reliability of the phenomenon of metal resistance is based on the following data:
   

   2.1.	Superresistance and polyresistance of the Antarctic microorganisms concerning six most toxic metals – Hg, Cu, Cr, Cd, Ni and Co.
   2.2.	Metal resistant microorganisms are widely occurred in all terrestrial biotopes in the investigated area of Antarctic Region that extents 60 kms from the North to the South and 30 kms from the West to the East.
   2.3.	High occurrence and the high concentration metal resistant microorganisms in typical Antarctic biotopes.
   2.4.	Ability of the Antarctic microorganisms to biogeochemical interaction with metals – i.e. they carry out reduction, sedimentation, mobilization and immobilization of toxic metals.

3. We suppose that metal resistant microorganisms could be "retro-forms" adapted to high concentrations of toxic metals in ancient times during neotectonic activity in Antarctic Region.

4. On the basis of superresistant Antarctic microorganisms new universal biotechnologies for purification of metal containing industrial waste water, and also biometallurgical technologies for extraction of metals from ore can be developed.

5. Studying the phenomenon of metal resistance Antarctic microorganisms is rather perspective as in theoretical and applied aspects, and demands the international cooperation for carrying out of complex research. 

We express sincere gratitude for support to scientists of the Chilean Antarctic institute to Dr. Rotamales, Dr. Veronike, the Chilean military Navy comandor first captain Arturo and to major Romano. 

We also thank our colleagues, Base Uruguay command Artigas and Russian wintering command from BELLINGSGAUSEN STATION, especially to Aleksey Shmaryn, for support during 13th seasonal Ukrainian Antarctic expedition. Slide 25.