Биологические науки/6. Микробиология

Khizhnyak S.V., Harlamova L.T., Voronov A.I.

Krasnoyarsk State Agrarian University, Russia

A new cave system in the Genevskaya Cave as a potential source of cold-adapted bacteria

Middle Siberian limestone caves are natural reservoir of cold-adapted bacteria and fungi [2, 3, 5, 6, 8]. These bacteria and fungi are of big interest for low-temperature biotechnology and for biological plant protection in boreal climate regions and in bioregenerative life-support systems based on higher plants [1, 4, 7].

The present research is devoted to the microbiological study of underground lakes in a new cave system "Zapadlo" (389 m length, 59 m depth) which was discovered in the Genevskaya Cave in 2007 and before an exploration was isolated from the main cave system with a glacier and a stone obstruction which prevented microorganisms penetration from the main cave system. The Genevskaya Cave (6000 m length, 90 m depth) also known as Zhenevskaya Cave is located in the Eastern Sayan Mountain area, Krasnoyarsk Krai, Russia. The cave was discovered in 1983 and explored in 1986-87. It is formed in the Early Cambrian limestone and has typical for Siberian caves characteristics: the temperature of air, water and cave sediments varies from 0 to +5ºC depending on the cave site with prevail temperature +4ºC; relative humidity of air is about 90-100%. Our previous researches demonstrated the presence of cold-adapted bacteria and fungi both in water and in cave sediments in the main cave system of the Genevskay Cave. The "Zapadlo" cave system has the same geological and climate characteristics as the main cave system.

Water samples were taken from small underground lakes located in three sites of "Zapadlo" cave system. Number of bacteria was calculated by colony count after cultivation at +7ºC and +35ºC on the following growth mediums: PD (peptone – 9,0 g/l; casein hydrolysate – 8,0 g/l; yeast extract – 3,0 g/l; NaCl – 5,0 g/l; Na₂HPO₄ – 2,0 g/l; agar – 20 g/l; pH = 7..7,5), Chapek (saccharose – 20,0 g/l; NH₄NO₃ – 2,0 g/l; KH₂PO₄ – 1,0 g/l; MgSO₄*7H₂O – 0,5 g/l; KCl – 0,5 g/l; FeSO₄ – trace quantity; agar – 20 g/l; рН = 6,5..7,0), Oligotrophic (25 ml of non-agarised PD and 25 ml of non-agarised Chapek and 20 g of agar per 1 liter of medium). Confidence intervals for numbers of bacteria were calculated using Puasson distribution.

Number of bacteria able to grow at low temperature varies from 5,0x10³ to 121,7x10³ of colony-forming units (CFU) per ml depending on the cave site and growth medium used for cultivation. Number of bacteria able to grow at temperature +35ºC varies from less then 0,1x10³ to 5,1x10³ CFU per ml (Tabl. 1).

Table 1 – Number of cultivated bacteria in water samples (CFU x10³)

Growth medium	Temperature of incubation
	+7ºC			+35ºC
	Average	Confidence interval (95%)		Average	Confidence interval (95%)
Site 1
Chapek	8,5	6,9	10,5	0,3	0,2	0,4
Oligotrophic	6,2	4,8	8,0	3,3	2,3	4,7
PD	5,0	3,8	6,6	<0,1*	–	–
Site 2
Chapek	121,7	115,1	128,7	<0,1*	–	–
Oligotrophic	69,5	64,5	74,9	2,1	1,3	3,3
PD	91,7	86,0	97,8	5,1	3,8	6,6
Site 3
Chapek	8,6	6,9	10,7	<0,1*	–	–
Oligotrophic	17,2	14,8	20,0	4,5	3,3	6,1
PD	12,6	10,6	15,0	<0,1*	–	–

* No colonies were formed at any dilution

Prevalence of bacteria unable to grow at high temperature (+35ºC) demonstrates that water bacterial community in the "Zapadlo" cave system is cold-adapted. From other hand, none of bacterial isolates are really psychrophilic in contrast with the main cave system of the Genevskaya Cave. Most of the cold-adapted bacteria and fungi previously isolated from the main cave system of the Genevskaya Cave are unable to grow at temperatures higher then 26ºC – 29ºC or even lower whereas the maximal growth temperature of strains isolated from the "Zapadlo" cave system lies between 30ºC and 35ºC. At temperature 35ºC these strains forms abnormal cells which subsequently die (Fig. 1).

Fig. 1 – Examples of abnormal cells of cave bacteria formed after incubation at high temperature (20-hour culture on agar slides, phase contrast microscopy): 1 – normal cells of strain MPA3 growing at +23ºC, 2 – normal cells of strain MPA6 growing at +23ºC, 3 – abnormal cells of strain MPA3 formed at +35ºC, 4 – abnormal cells of strain MPA6 formed at +35ºC

According to their cultural characteristics and growth temperature range the bacteria isolated from the "Zapadlo" cave system differ from the strains isolated from the main cave system of the Genevskaya Cave what can be explained with long time isolation of the "Zapadlo" from the main cave system. Despite an absence of real psychrophiles the "Zapadlo" cave system is of interest for search of strains for biological plant protection in bioregenerative life-support systems because their inability for growth at human body temperature makes them safety for humans.

References:

1. Ilients, I.R. Cave communities of micromycetes as a source of strains for agricultural and environmental biotechnology: PhD thesis. Krasnoyarsk State Agrarian University, Krasnoyarsk, 2011: 140 pages (in Russian)

2. Khizhnuak, S., Tausheva, I., Berezikova, A. Supposed contribution of cave microorganisms to a karstic process in the limestone caves of Easten Siberia. Contributions to the IGCP448 meeting in Bowling Green, Ky, USA, June3-6 2003, Part III-1 World comparison of karst ecosystem. Bowling Green, 2003: 99.

3. Khizhnuak, S.V., Tausheva, I.V., Berezikova, A.A., Nesterenko, E.V. Ecological and biological peculiarities of microorganisms from underground karst cavities of the Middle Siberia. Homeostasis of forest ecosystems: Proc. X International Symposium "Concept of Homeostasis: Theory, Experiments and Application". Novosibirsk, 2001: 161-165.

4. Khizhnyak, S.V., Lankina, E.P., Ilients, I.R. Estimation of effectiveness of cold-adapted cave microorganisms in biological control of common root rot of cereals. Vestnik Krasnoyarsk State Agrarian University, 2009, 6: 49-52. (in Russian)

5. Khizhnyak, S.V., Nesterenko, E.V., Berezikova, A.A., Trusey, I.V. The microbial communities of Middle Siberian cold caves. 3-rd International Workshop on Ice Caves Kungur Ice Cave, Perm Region Russia, May 12-17: Volume of Abstracts. Kungur, 2008: 56-57.

6. Khizhnyak, S.V., Tausheva, I.V., Berezikova, A.A., Nesterenko, Y.V., Rogozin D.Y. Psychrophilic and Psychrotolerant Heterotrophic Microorganisms of Middle Siberian Karst Cavities. Russian Journal of Ecology, 2003, 34 (4): 231-235.

7. Lankina, E.P. Cave bacterial communities as a source of strains for biological protection of plants against diseases: PhD thesis. Krasnoyarsk State Agrarian University, Krasnoyarsk, 2011: 140 pages (in Russian)

8. Nesterenko, E.V. Micromycetes of Middle Siberian Karst Cavities: PhD thesis. Krasnoyarsk State Agrarian University, Krasnoyarsk, 2007: 134 pages (in Russian)