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Biology Bulletin, Vol. 32, No. 6, 2005, pp. 549–555. Translated from Izvestiya Akademii Nauk, Seriya Biologicheskaya, No. 6, 2005, pp. 664–671.Original Russian Text Copyright © 2005 by Bel’kova, Parfenova, Suslova, Ahn, Tazaki.

INTRODUCTION

Hydrothermal vents feature certain extreme param-eters including high temperature and mineralization orsometimes elevated concentrations of toxic and heavymetals such as cadmium, arsenic, mercury, and iron.Aquatic microbial community and bacterial mats estab-lished around hydrothermal vents are a unique subjectfor both microbiological and geological studies. Micro-biologists isolate and describe new species of thermo-philic microorganisms with unique enzyme propertiesand metabolism (Gonzalez and Robb, 2000; Prokofevaet al

., 2000). These microorganisms are involved in avariety of transformations of elements includingvalency conversions (oxidation and reduction) or phys-ical transitions (solid, liquid, and gas). Many of thesetransformations are key stages in geochemical pro-cesses going in hydrothermal ecosystems. The presenceof modern biogenic minerals forming de novo

at hydro-thermal vents has been shown previously (Ferris et al

.,1986, 1987; Ehrlich, 1996; Fortin et al

., 1998; Bonnyand Jones, 2003; Belkova et al

., 2004; etc.). Biogenicformation of laminated minerals, such as iron-manga-

nese concretions and stromatolites, as a result of bio-geochemical processes going in hydrothermal ecosys-tems is of apparent interest (Tazaki, 1999; Miyata et al

.,2001; Brake et al

., 2002; etc.). Hence, bacterial matsforming at hydrothermal vents are a unique naturalcommunity of microorganisms realizing complexgeochemical transformations and the enzyme activitiesof these bacteria are of great interest.

The goal of this work was to study the compositionof bacterial mats formed at the Kotelnikovsky Hot

Springs and to test cultured thermophilic microorgan-isms inhabiting this hydrothermal ecosystem for poten-tial enzyme activities such as phosphatase, proteinase,lipase, amylase, and lecithinase.

MATERIALS AND METHODS

Sampling and field operations.

Samples of water,mineral deposits, and biomats were collected in July

2001, August 2002, and September 2003 from theKotelnikovsky Hot Springs at the northwestern coast of Lake Baikal (Fig. 1). Bacterial mats were fixed imme-diately with glutaraldehyde (final concentration of 2.5%) and stored at 4

°

C until microscopic analysis.Hot-spring water properties such as temperature, pH,redox potential (Eh), electrical conductivity (EC), anddissolved oxygen concentration (DO) were measuredin the field using portable EC/pH meter WM-22EP(TOA, Japan) and Eh-meter (Horiba, Japan).

Chemical analysis.

Unfixed samples of mineraldeposits and bacterial mats were air dried at room tem-perature and ground to fine powder. The samples were

analyzed using spectrophotometry (JEOL JSX 3201,Japan) with Rh K

α

and an accelerating voltage of 30 kV under vacuum.

Scanning electron microscopy.

Samples werefreeze-dried as described elsewhere (Belkova et al

.,2004). The bacterial mat samples fixed with 2.5% glut-araldehyde were postfixed with t-butyl alcohol, frozen,and dried under low vacuum in the scanning micro-scope chamber. The samples were mounted on copperstubs with a double-sided carbon tape, carbon coated,

Biodiversity and Activity of the Microbial Communityin the Kotelnikovsky Hot Springs (Lake Baikal)

N. L. Bel’kova*, V. V. Parfenova*, M. Yu. Suslova*, T. S. Ahn**, and K. Tazaki***

* Limnological Institute, Siberian Division, Russian Academy of Sciences, ul. Ulan-Batorskaya 3,P.O. Box 4199, Irkutsk, 664033 Russia

** Department of Environmental Science, Kangwon National University, Chunchon, 200-701 Korea

*** Department of Earth Sciences, Faculty of Science, Kanazawa University, Kanazawa, 920-1192 Japan

e-mail: [email protected]

Received December 2, 2004

Abstract

—Complex microbiological and chemical analyses of water and bacterial mats were performed in theKotelnikovsky Hot Springs (Lake Baikal). Transmission electron microscopy demonstrated that short rodsabout 1.2–2 µ

m in diameter predominated in the natural microbial community. Scanning electron microscopycoupled with chemical analysis revealed a characteristic P peak in the bacteria-like mineral particles, whichsuggests their biogenic origin. Most strains of the thermophilic microorganisms were gram-positive spore-

forming rods and can be assigned to the genus Bacillus

. Assays for potential enzyme activity demonstrated thatmost of the strains tested were active at high temperature. The data obtained suggest high activity of the bacte-rial community in situ

and its particular role in the functioning of the hydrothermal ecosystem.

MICROBIOLOGY

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and observed with scanning electron microscopeequipped with an energy-dispersive spectrometer(SEM: JEOL-JSM-5200LV; EDX: Philips-EDAXPV9800 STD, Japan).

Transmission electron microscopy and diffractionanalysis.

Bacterial cells were concentrated in an aliquotof thermal water and fixed with 1% glutaraldehyde. Thecell suspension without preliminary contrasting wasmounted on microgrids and air-dried. Whole bacterialcells and mineral particles were examined under atransmission microscope (JEOL JEM-2000 EX, Japan)at an accelerated voltage of 80 to 120 kV at differentmagnifications. In addition, electron diffraction analy-sis was carried out to describe the mineral compositionof the particles.

Isolation and growth of thermophilic bacteria.

Batch cultures of thermophilic microorganisms weregrown on the following selective media. Medium H:

glucose, 1 g/l; tryptone, 5 g/l; yeast extract, 2.5 g/l;

ëaël

2

·

2H

2

O, 0.57

g/l (Shooner et al

., 1996); mediumJXT: yeast extract, 1 g/l; tryptone peptone, 1 g/l;

Na

2

S

2

O

3

·

5H

2

O, 1

g/l, pH was adjusted to 7.0 with HCl(Sako et al

., 1996); medium MBM: polypeptone, 5 g/l;yeast extract, 1 g/l; K

2

HPO

4

, 6 g/l; KH

2

PO

4

,

2

g/l;

MgSO

4

·

7H

2

O

,

0.5

g/l; L-tyrosine, 0.5 g/l (Sung et al

.,2002); medium NB: broth for microorganism cultiva-tion (NNPTs GIP, Russia), 8 g/l (Beffa et al

., 1996). Analiquot of thermal water or bacterial mat (1 ml) was

added to tubes with liquid media H, JXT, MBM, or NBand incubated for 3 to 7 days at 53

°

C. Individual colo-nies were isolated by inoculating 1 ml of the batch cul-ture to the corresponding solid medium containing1.5% gelrite (Duchefa Biochemie, Netherlands) and

incubated for 7 to 14 days at 53

°

C.

Enzyme activity assay. Phosphatase

activity wasdetermined using a standard kit Alkaline PhosphataseFL (Vital Diagnostics SPb, Russia). The reagent con-taining p-nitrophenyl phosphate (0.25 ml) was added toeach well of plates together with the studied cultures (1loop). Phosphatase activity was determined by theintensity of yellow staining of the resulting suspensionafter incubation for 1 h at room temperature and 24 h at50

°

C.

Proteinase

activity was assayed on milk agar con-taining defatted milk in 1.8% starvation agar (15 ml per100 ml).

Lecithinase (phospholipase C)

activity was deter-mined using thoroughly suspended chicken egg yolk.The medium was poured into sterile Petri dishes and thestudied cultures were stab-inoculated. A clearance zonewas observed in the presence of proteolytic or lecithi-nase activity around stabs. Results were recorded afterincubation for 3 days at room temperature and 2 days at50

°

C.

Amylase

activity was determined on starch agar(KH

2

PO

4

, 0.5 g/l; K

2

HPO

4

, 0.5 g/l; MgSO

4

· 7H

2

O,

53°

106°

40 km

ARCTIC OCEAN

Siberia KotelnikovskyHot Springs

N

Baikal

Lake

Irkutsk

Mongolia

ChinaJapan

Irkutsk

A n g

a r a

S e l e n

g a

Fig. 1.

Map of the Kotelnikovsky Hot Springs, northwestern coast of Lake Baikal.

B a i k a

l L a k

e

Listvyanka

Settlement

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BIODIVERSITY AND ACTIVITY OF THE MICROBIAL COMMUNITY 551

0.2 g/l; (NH

4

)

2

SO

4

, 0.2 g/l; starch, 10 g/l, and agar,15 g/l) (Rodina, 1965) sterilized and poured into sterilePetri dishes. Tested cultures were stab-inoculated andincubated for 3–5 days. Starch hydrolysis was visual-ized as a clearance zone around stabs after staining withLugol’s solution.

Lipase

activity was determined on 20 g/l tributyrinagar (Fluka no. 91015). After sterilization at 121

°

C for

15 min, the medium was cooled to 80

°

C, comple-mented with tributyrin (Fluka no. 91010), stirred on amagnetic stirrer for 2–3 h until complete homogeniza-tion, and poured into Petri dishes. Tested cultures werestab-inoculated and incubated for 5 days at 50

°

C.Clearance zones were indicative of lipase activity.

RESULTS AND DISCUSSION

Chemical analysis of thermal water, bacterial mats,and mineral deposits.

Field measurements of physico-chemical parameters (temperature, pH, Eh, EC, anddissolved oxygen) of thermal water demonstrated:alkaline pH (8.9), reducing conditions (–330 mV), lowdissolved oxygen level (2.7 mg/l), and high tempera-ture (76

°

C). The thermal water can be assigned to mod-erately mineralized (490 µ

S/cm). Published data indi-cate that the Kotelnikovsky Hot Springs have a fluo-ride–carbonate–sodium type of water with the totalmineralization of 0.32 g/l and silicate concentration of 120–130 mg/l (

Baikal. Atlas

, 1993). Chemical analysisof bacterial mats and mineral deposits demonstratedthat the deposits formed under natural conditions athydrothermal vents largely included silicon and cal-cium with insignificant contents of sulfur and iron.Bacterial mats contained, apart from high concentra-tions of silicon and calcium, biogenic elements such as

magnesium, phosphorus, sulfur, potassium, and man-ganese. In addition, very high concentrations of iron aswell as aluminum and titanium were observed in thebiomats. The obtained data indicate selective accumu-lation of elements such as iron and calcium in bacterialmats. Previously, amorphous phases of silica, calcite,and fluorite were identified in mineral deposits and bac-terial mats from the Kotelnikovsky Hot Springs by X-ray diffraction analysis (Belkova, 2004; Belkova andTazaki, 2004). In addition, the biogenic provenancewas proposed for the mineral forms by analysis of thesamples by transmission microscopy (Belkova andTazaki, 2004) and a particular role of microorganismsinhabiting the hydrothermal bacterial mats was denoted

in the precipitation of silicates and coprecipitation of calcium in minerals such as calcite and fluorite (Belk-ova, 2004).

Microscopic analysis of microbial community of theKotelnikovsky Hot Springs.

Microbial community of thermal water was analyzed by TEM. Short rod-shapedbacteria about 1.2–2 µ

m in length proved to be thedominant morphological form. Mineral particles pre-cipitated on their cell surface (Fig. 2). Electron diffrac-tion analysis demonstrated the presence of amorphous

particles alone, suggesting the initial stages of mineralstructure formation. These particles seem to attach toexopolysaccharides detectable on the cell wall surface(Beveridge, 1989; Schultze-Lam et al

., 1996; Fortin

et al

., 1997; Belkova, 2004). Chemical analysis of themineral particles demonstrated the presence of siliconand calcium.

A variety of single-cell algae and microorganismswas revealed in the bacterial mats of the KotelnikovskyHot Springs. Most of them were aggregated with min-eral particles, some of which can have a biogenic origin(Figs. 3a, 3b). Chemical analysis of the mineral parti-cles (Fig. 3b) showed strong peaks of silicon and cal-cium with traces of Mg, Al, P, S, and Fe. A low but typ-ical peak of phosphorus identified in spherical particleswith a diameter of about 0.5 µ

m points to their biogenicorigin.

Isolation of thermophilic microorganisms and assay for their enzyme activities.

The collection of thermo-philic microbial cultures included 39 strains isolated

from water and bacterial mats of the Kotelnikovsky HotSprings. Growth of thermophilic microorganisms wasobserved on all tested media (table). Overall, the high-est number of cultures was isolated on NB medium(13 strains) composed of broth alone. Eleven strainswere isolated on MBM medium containing K

2

HPO

4

,KH

2

PO

4

, MgSO

4

, and amino acid L-tyrosine in addi-tion to nutrients (polypeptone and yeast extract). Thelowest number of strains (8 and 7) was obtained on Hand JXT media, respectively. These media include tryp-

200 nm

Fig. 2.

Transmission electron micrograph of bacteria iso-lated from water of the Kotelnikovsky Hot Springs; arrowsindicate amorphous mineral particles attached to the cellwall surface.

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2.0 4.0 6.0 8.0keV

0

Si

Al PS FeMgCaK

β

CaK

α

(b)

(a) 5 µ

m

1 µm

Fig. 3. Scanning electron micrographs of a diatom (a) and bacteria-like mineral particles (b) revealed in bacterial mats of theKotelnikovsky Hot Springs; inset to (b) chemical spectrum of the particles, whose location is indicated by an arrow.

tone, yeast extract, and low salt admixtures. At the sametime, most thermophilic bacteria were isolated fromwater samples on MBM medium (7 out of 11). MediumH is favorable for microorganisms from bacterial mats:

6 and 2 strains were isolated from bacterial mats andwater samples, respectively. Similar numbers of strainsfrom bacterial mats and water samples were isolated onNB and JXT media (table). Hence, the composition of selective medium used to grow and isolate pure culturesof thermophilic microorganisms influences the rates of their survival and adaptation to growth under laboratoryconditions. Morphological analysis demonstrated thatmost of thermophilic bacterial cultures isolated onthese media are gram-positive spore-forming rods and

can be assigned to the genus Bacillus. In addition, two

yeast strains were isolated on MBM medium (table).

Both the greatest number and diversity of bacterial

forms were obtained on NB medium despite its simple

composition. Four out of 13 isolated strains were gram-negative rods; 8, gram-positive rods; and one culture

was gram-variable (table). The absence of spores was

typical for all strains isolated on NB medium. Analysis

of the cultivation results demonstrated that complete

description of natural community of thermophilic

microorganisms requires their isolation on selective

media with different nutritional and mineral composi-

tion.

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Enzyme activities of thermophilic bacteria isolated from water and bacterial mats of the Kotelnikovsky Hot Springs, north-western coast of Lake Baikal

S t r a i n Source Medium

Morphology Phosphatasea Protein-

ase, mmd

t 1 /

Amy-lase, mm

t 2

Lipase,mmt 2

Lecithi-nase, mm

t 1 / t 2Gram

stainingCell

shapeSpores 1 hb, t 1 24 hb, t 2

1 2 3 4 5 6 7 8 9 10 11 12

1 Water JXT + Small rods – – ++ –/– – – –2 Water JXT + Long rods – +++ +++ 3/11 12 4 4/19

3 Water JXT + Rods + + +++ 3/11 17 4 6/20

4 Biomats JXT – Rods – + +++ –/– 25 – –/–

5 Biomats JXT + Rods + ++ +++ 5/12 – 4 4/15

6 Biomats JXT + Rods + – ++ 6/13 – 4 4/15

7 Water JXT + Rods + ++ +++ 5/12 11 + 6/17

8 Biomats MBM + Rods + – + 18/30 2 12 7/11

9 Biomats MBM + Rods + – + 20/30 2 12 7/11

10 Biomats MBM + Rods + – +++ 4/12 2 8 6/16

11 Biomats MBM + Rods + – + 20/32 – 13 8/16

12 Water MBM + Yeast – – +++ 8/16 – 6 3/9

13 Water MBM + Yeast – – +++ 6/15 – 4 4/814 Water MBM + Rods + ++ +++ 9/18 – 6 +/8

15 Water MBM + Rods – + +++ 8/16 3 4 7/7

16 Water MBM + Rods – +++ +++ 2/10 – 6 4/12

17 Water MBM + Rods + – ++ 5/10 – 5 5/12

18 Water MBM + Rods + – +++ 6/15 – – 6/12

19 Biomats NB + Rods – – – –/– – 4 6/8

20 Biomats NB – Rods – + + –/– – 3 –/–

21 Biomats NB + Large rods – – – –/– – 1 11/11

22 Biomats NB + Large rods – – – –/16 – 8 8/10

23 Biomats NB +/– Large rods – – – –/17 – 5 8/11

24 Biomats NB + Small rods – – ++ 25/33 9 – 6/10

25 Water NB + Large rods – – – –/9 – 3 –/–26 Water NB + Rods – +++ +++ –/13 – 20 4/6

27 Water NB + Rods – + +++ 6/12 – 20 4/6

28 Water NB + Small rods – – ++ –/– – – –/–

29 Water NB – Rods – – + –/– – – –/–

30 Water NB – Small rods – – + –/– – – –/–

31 Biomats NB – Rods – – ++ –/– – – –/–

32 Biomats H + Rods + +++ +++ 5/10 – 20 5/7

33 Water H + Large cocci – – – –/10 – 7 –/6

34 Biomats H + Rods – – – –/6 – 7 –/–

35 Biomats H + Rods + – – 5/26 – 15 12/17

36 Biomats H + Small rods + +++ +++ 6/26 – 20 5/8

37 Water H + Rods + + +++ –/6 10 11 –/–

38 Biomats H + Rods + ++ +++ –/15 15 14 –/–

39 Biomats H + Rods + ++ +++ –/10 – 17 –/–

Total number of strains with enzyme activity: 16 31 21/30 11 31 26/27

Proportion of strains with enzyme activity in total numberof studied strains, %

41 79 54/77 28 79 67/69

a Phosphatase activity was evaluated by the intensity of yellow stain: (+++) bright yellow; (++) yellow; (+) light yellow; (–) no staining.b Incubation time.c t 1, 20°C; t 2, 50°C.d Proteinase, amylase, lipase, and lecithinase activities were determined from clearance zone size, mm.

t 2c

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All isolated strains of thermophilic microorganismswere tested for the presence of enzyme activities,namely, phosphatase, proteinase, lecithinase, lipase,and amylase (table). A high proportion of strains dem-onstrated some enzyme activity. Phosphatase activitywas observed in 79% (31 strains) of total number of analyzed strains; 50°C was optimal for most these cul-tures. As low as 16 out of 39 strains (41%) demon-

strated alkaline phosphatase activity after growth atboth 20 and 50°C. Moreover, only 6 out of 16 strainsdemonstrated evenly high activity at both temperatures(+++), while other 15 strains demonstrated this activityonly after incubation at 50°C (table). Medium compo-sition clearly influenced the capacity of strains for par-ticular enzyme activities. For instance, all strains iso-lated on JXT and MBM media had phosphatase activityafter incubation at 50°C. On the other hand, onlyslightly over 60% of total number of strains isolated onNB and H media demonstrated this enzyme activity (61and 62%, respectively).

Proteolytic activity was determined after incubationat 20 and 50°C. Note that the clearance zones variedfrom 3 to 25 mm at room temperature, while it rangedfrom 6 to 33 mm at 50°C. As low as 21 strains (54%)demonstrated the activity after incubation at 20°C,while 30 strains (77%) demonstrated catalytic activityat 50°C. This indicates that 50°C is the optimal temper-ature for growth and metabolism of thermophilic bacte-ria from these springs.

Quite similar numbers of tested strains (67 and69%) demonstrated lecithinase activity after growth at20 and 50°C. Similar to proteolytic activity, clearancezones were observed in all strains isolated on MBMmedium (10 in total).

Lipase activity was determined at 50°C. A total of 31 strains (79%) demonstrated the capacity to hydrolyzetributyrin substrate. The clearance zones ranged from 3to 20 mm. Only two out of eight strains with lipase activ-ity isolated on NB medium demonstrated clearancezones up to 20 mm, otherwise it ranged from 1 to 8 mm.High lipase activity was observed for the strains isolatedon H medium (8 strains): the clearance zones varied from7 to 20 mm. A much lower lipase activity was observedin four strains isolated on JXT medium; the correspond-ing clearance zones were 1–4 mm.

The strains demonstrated the lowest starch-hydro-lyzing (amylase) activity; various clearance zones wereobserved in as low as 11 out of 39 tested strains (28%).

Note that the zone size differed for strains isolated ondifferent media. For instance, the greatest clearancezones (from 11 to 25 mm) were observed for strainsisolated on JXT medium (4 strains) containing yeastextract, tryptone peptone, and Na2S2O3. The strains iso-lated on MBM medium (4 in total) demonstrated smallclearance zones not exceeding 2–3 mm. As low as 1 and2 strains isolated on NB and H media, respectively,demonstrated amylase activity, although the clearancezones were 9 and 10–15 mm, respectively. Hence, most

strains of thermophilic microorganisms demonstratedhigh phosphatase, proteolytic, lipase, and lecithinaseactivities, while amylase activity was observed in theirminor fraction. Note that the composition of mediaused to isolate thermophilic microorganisms influencestheir enzyme activities. All strains isolated on MBMmedium demonstrated phosphatase, proteinase, andlecithinase activities and 91% of them had lipase activ-

ity as well. Despite their high number, the strains iso-lated on NB medium demonstrated the lowest enzymeactivity.

CONCLUSIONS

Physicochemical analysis of water from theKotelnikovsky Hot Springs has confirmed the previousdata on moderate mineralization, high temperature, andlow oxygen concentration. The biomats and mineraldeposits contained high concentrations of silicon andcalcium; in addition, magnesium, phosphorus, potas-sium, manganese, sulfur, aluminum, and titanium werefound in them. Despite extreme natural conditions, cul-turable thermophilic microorganisms with high poten-tial enzyme activity inhabit the water and biomats.Analysis of the obtained data on growth of studiedmicroorganisms demonstrated considerable depen-dence of studied activities on the composition of cul-ture media. In addition, growth and assay temperaturehad a notable impact on potential enzyme activity of isolated strains: the activity was higher after incubationat 50 than 20°C.

The obtained data suggest that water and bacterialmats of the Kotelnikovsky Hot Springs are inhabited bya unique community of thermophilic microorganisms.This bacterial community clearly plays a particular role

in the functioning of the hydrothermal ecosystem,which includes the biogeochemical transformationsmediated by their high metabolic activity.

ACKNOWLEDGMENTS

This work was supported in part by the JapaneseMinistry of Education, Science, and Culture; Govern-ment of Korea (MG 02-0101-002-2-2-0), Presidium of the Russian Academy of Sciences (25.5 “Evolution of Silicon Biomineralization Systems”), Program forBasic Research of the Russian Academy of Sciences(subprogram 13.19), and Russian Integration Project of the Siberian Branch and Far East Division of the Rus-sian Academy of Sciences (no. 58).

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