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Analele ştiinţifice ale Universităţii “Al. I. Cuza” IaşiTomul LII, s. II a. Biologie vegetală, 2006

CHARACTERIZATION OF THE MICROBIOTA FROM DIFFERENT TYPES OFANTHROPOGENIC SOILS

SIMONA DUNCA, C. TĂNASE*, M. ŞTEFAN*, ANA COJOCARIU

Abstract: The paper present the quantitative analysis of microbiota of different types of soils fromantropized zones of Iasi and the macro- and micro-morphological characterization of isolated strains.Key words: microbiota, soil, anthropogenic

Introduction

The soil group together all the bacteria implicated in the circuit of carbon,nitrogen, sulphur, iron and of the other element from environment. The species of generaAcinetobacter, Agrobacterium, Arthrobacter, Bacillus, Brevibacterium, Cellulomonas,Chromobacterium, Pseudomonas, Sarcina, Staphylococcus and Xanthomonas are foundconstantly in different types of soils [2].

The structure of the soil represents a good indicator for the biologic activity, assoil aggregates define the most important microhabitats for microorganisms. The soilshould have enough pores, filled with gases, in order to allow the gas exchange with theatmosphere, reducing thus the risk of formation of the anaerobic area. Within theaerobiosis, the activity of microorganisms is much more intense, and the mineralizationprocesses take place in optimal conditions [1].

The development of microorganisms is conditioned by the soil pH. Generally, thesoil pHs vary between 4.5 and 8.5. Most microorganisms prefer soils with neuter pH(between 6 and 7), as, under these conditions, the capacity for using nutrients is maximal.For instance, actinomycetes develop better at a neuter pH and do not tolerate well the acidpH. Nevertheless, in the soil, microorganisms can develop for pH in the range between 1and 13 [6].

Temperature is a variable factor between the layers from the soil surface,presenting diurnal variations, with rapid decreases at night and season variations accordingto latitude and altitude. The temperature increases and variations are influenced by thewater content and by depth, and they are higher in the soils where there is no vegetation[7].

Aeration is the process by which gases produced or consumed under the terrestrialsurface are exchanged with the gases in the atmosphere air [11]. Ideally, a well aerated soil

'Al. I. Cuza' University of Iaşi, Faculty of Biology, B-dul Carol I no. 11, 700506 – Iaşi, Romania Anastasie Fătu' Botanical Garden of Iaşi, 7-9 Dumbrava Roşie Str., 700487 – Iaşi, Romania

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contains enough oxygen for the respiration of the roots of plants and aerobicmicroorganisms, which oxygenate organic compounds to CO2. Depth is a secondaryecological factor.

The number of bacteria is lower at the soil surface, because of the bactericidaleffect of solar radiations, which is maximal in the first 5 centimetres under the surface andit decreases gradually with depth [8].

The optimal humidity level for the activity of aerobic bacteria is estimated at waterretention capacity of 50-70%; the oscillations of humidity determine variations of thebacteria community [9]. Related to the soil type, researches show that the highest numberof bacteria is present in black soils (3.6 x 106 / g of soils), but also in podsoils (2.1 x 106 / gof soils), grey soil and peat [11].

Material and methods

The isolation of microorganisms was made by soil dilutions suspension in sterilewater, using a dilution coefficient in the range of 10. For each dilution in the range of 10obtained, 1 ml of suspension was placed (by display) on 3 Petri plates, and then an averagewas made for the developed colonies. The plates were incubated in the thermostat at 28°Cof temperature, for seven days. In order to reduce the risk of calculation errors, final resultswere expressed in colony-forming units (CFU).

The number of CFU in a gram of sample is calculated according to the nextformula [3]:

CFU / gram of soil = a x 10n /Vwhere: a – number of colonies;10n – dilution;V – volume of inoculum.After seven days of incubation, several strains were isolated and numbered on the

surface of the colony-developing plates. In order to obtain pure cultures, isolated cultureswere previously verified. For this purpose, all cultures were examined microscopically inorder to check their purity and to be able to observe the morphology of bacterial cells.Microscope examination supposed realizing smears and colouring them by the Gramstaining technique. After colouring, strains were kept at 4°C of temperature, on freshculture medium [10].

In order to characterize isolated strains, we took into consideration the macro- andmicro-morphological description. The macro-morphological description consisted incharacterizing microorganism colonies, using the following criteria: colony type, size,shape, edge aspect, colony profile, consistence, transparency/opacity and colour. For thedescription of the micro-morphological characteristics of the microorganisms isolated,smears were made from the pure cultures obtained, which were coloured by the Gram

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method. Smears were microscopically examined with the immersion objective, observingthus the peculiarities of the microscopic aspect of microorganisms [4].

The samples results from different anthropogenic zones of Iasi, being noted asfollowing: 1a and 1b for samples from 'Anastasie Fatu' Botanical Garden, 2a, 2b, 3a, 3b, 4a,4b, 5a, 5b, 6a, 6b, 6c, 7a, 7b, 7Aa from SC Antibiotice SA, 7y from CUG, 8 and 9 fromTomesti, 10i, 10ii, 10iii, 10iv from CET Holboca.

In order to quantify isolated strains, an identification index was added to each soilsample (ex. 1a1).

Results and discussions

The results of the researches concerning the quantitative analysis of the microbiotapresent in the soil samples analyzed (Table I) indicate that the highest microbial load wasidentified in the ash samples of the pit coal from CET Holboca (10 iii and 10 iv), whichregistered 91.9 x 107 CFU/g of soil, and 53.2 x 106 CFU/g of soil, but also in the (5b)sample from SC Antibiotice SA (41.5 x 107 CFU/g of soil). A lower microbial load wasregistered in the soil samples (2a and 6c) collected from SC Antibiotice SA, whichregistered a number of 50 x 103, and 26.2 x 104 CFU/ g of soil, respectively.

By classifying the types of soils analyzed, according to the abundance of microbialcommunities, we can make a difference between:

- soils where the number of microorganisms is relatively close between the twolayers (samples from 'Anastasie Fătu' Botanical Garden);

- soils where the number of microorganisms is higher in the deep layer (soil fromSC Antibiotice SA and pit coal from CET Holboca);

- soils where the number of microorganisms is higher in the superficial layer (soilfrom SC Antibiotice SA).

The number of microorganisms present in the superficial layers of the soil candecrease with depth; this could be explained by the rhythmical accumulation and thedecomposition of toxins in the soil. Taking into consideration the influence of the aerationon the biological activity that takes place in the soil, it is known that this activity decreaseswith depth, together with a relative growth of anaerobic processes. The decrease of thenumber of microorganisms in the soil can also be caused by the nutrition conditionsmicroorganisms find in the soil, which are more favourable in the surface layers, due to thegreater humus quantity and to a more frequent feed with organic residues. The soil samplescollected in the Botanical Garden presented a high content of organic carbon and humus;this led to the identification of a relatively high load (56 x 104, and 44 x 104 CFU/ g of soil,respectively). There were some soil samples with a lower microbial load in the surfacelayers; this could be also caused by the bactericidal action of light, especially of radiationsin the blue-violet spectrum, which determine peroxide formation or protein hydrolysiswithin the microbial cells. UV radiations can also have a strong bactericidal influence

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(especially in the wavelengths range of 2000-3000 Ǻ), but their effects are negligible,taking into account that they have a reduced power when entering the soil.

Based on the direct influence of the soil structure on the distribution ofmicroorganisms into the soil, there were quantitative variations in the types of soilanalyzed, decreasing in number from entianthroposoil to cambic chernozem, phaeziom andpreluvisoil.

Table I - Microbiota quantitative analysis in different soil samples

Analyzed sample CFU/g soil

1 a 56 x 104

1 b 44 x 104

2 a 50 x 103

2 b 22,2 x 106

3 a 32 x 104

3 b 60 x 103

4 a 23,2 x 104

4 b 57 x 103

5 a 23,7 x 105

5 b 41,5 x 107

6 a 87 x 104

6 b 35,9 x 104

6 c 26,2 x 104

7 a 14 x 105

7 b 83 x 103

7 Aa 11,4 x 104

7 y 58 x 104

8 74 x 105

9 18,9 x 105

10 i 31,4 x 105

10 ii 52 x 103

10 iii 53,2 x 106

10 iv 91,9 x 107

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As result of our researches we have isolated in pure cultures 67 strains ofmicroorganisms, grouped in two great taxonomic groups: bacteria (55 strains, representing82% of the total strains isolated) and actinomycetes (12 strains, representing 18% of thetotal strains isolated) – Fig. 1. The isolation of the actinomycetes strains, especially in thesoil samples collected from the SC Antibiotice SA dust hole (4-7) can be correlated withthe weakly alkaline pH of these types of soil, favourable to the development of the group ofmycelian bacteria. We compared the results of our research with the information in thespecialty literature, which states that actinomycetes represent approximately 20% of thesoil microbiota, and our researches confirm it.

82%

18%

Bacteria Actinomycetes

Fig. 1 – Percentage representation of isolated microorganisms

Isolated strains were characterized from a macro-morphological point of view(Table II); after the analysis of different types of colonies, it was noticed that most isolatedmicroorganisms form smooth “S”-type colonies and rugose “R”-type colonies and only afew species form mucous colonies. Colony size varies from 1-2 mm to large colonies (4.6cm). Colony shape can be round or irregular (for most strains), or filamentous. Weidentified mainly a whole or irregular aspect for colony edge, and there were few cases ofdenticulate or lobate edge. Isolated colonies presented different profiles, from flat to round,raised, hemispheric, convex or crater-shaped. Colonies had a mucilaginous or dryconsistence. As to transparency/opacity, it was noticed that most strains form opaquecolonies, weakly pigmented, a characteristic of microorganisms present in the soil (Photos1a, 1b, 1c, 1d).

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Table II. Macro-morphological aspect of bacterial strains isolated from soil samples

Sample Isolatedstrains

Colonytype Size Shape Edge

aspects Profile Consistence Transparency/Opacity Color

1a1 R 1,2 cm round lobate flat mucilaginous opaque white

1a2 S 5 mm round whole flat mucilaginous opaque grey

1a3 S 8 mm round whole umbonated mucilaginous opaque white1a

1a4 S 3 mm round whole flat mucilaginous opaque yellow

1b1 S 3,5 cm irregular lobate flat dry opaque white

1b2 R 6 mm irregular lobate hemispheric mucilaginous opaque grey

1b3 S 9 mm round whole hemispheric mucilaginous opaque white1b

1b4 S 6 mm round whole umbonated dry opaque yellow

2a1 R 3,5 cm irregular lobate flat dry opaque white

2a2 S 5 mm round whole convex mucilaginous transparent yellow

2a3 S 7 mm irregular whole hemispheric mucilaginous opaque yellow2a

2a4 S 6 mm round whole flat dry opaque white

2b1 S 3 mm round whole hemispheric mucilaginous opaque orange

2b2 R 8 mm round whole umbonated dry opaque actinomycetes withwhite aerian micelia2b

2b3 S 6 mm round whole flat dry opaque cream-colored

3a1 R 4,6 cm irregular lobate flat dry opaque white3a

3a2 S 6 mm round whole umbonated mucilaginous opaque yellow

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3a3 S 8 mm round whole hemispheric mucilaginous opaque white-rosy

3a4 S 7 mm irregular lobate umbonated mucilaginous opaque cream-colored

3b1 R 1,5 cm irregular serrated crateriform mucilaginous transparent cream-colored

3b2 S 1cm round whole hemispheric mucilaginous opaque white-rosy

3b3 S 4 mm round whole raised mucilaginous opaque yellow

3b4 R 1,1cm irregular lobate flat dry opaque white

3b

3b5 S 1 cm round whole convex mucilaginous opaque white-rosy

4a1 S 8,3 mm round whole flat mucilaginous transparent yellow

4a2 R 7 mm irregular lobate flat dry opaque white

4a3 S 3 mm irregular whole raised dry opaqueactinomycetes withwhite-rosy aerian

micelia, center white,downy

4a4 S 6 mm irregular whole hemispheric slime opaque blue- greenish

4a

4a5 M 7 cm filamentous serrated flat dry transparent white

4b1 M 3 cm irregular whole raised mucilaginous opaque limits cream-coloredwith blue pigment

4b2 R 3mm irregular whole raised dry opaque actinomycetes withscant aerian micelia

4b

4b3 S 1,5 cm round whole flat dry transparent white

5a1 R 7 mm irregular irregular flat dry opaque yellow5a

5a2 R 1,3 cm irregular lobate flat mucilaginous transparent milky-white

5b1 R 1,7 cm round serrated flat dry opaque milky-white

5b2 R 1,4 cm irregular lobate flat dry opaque white

5b

5b3 S 4 mm round whole flat mucilaginous opaque white

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5b4 S 3 mm round whole raised mucilaginous opaque orange

6a1 S 1,8 cm round whole raised mucilaginous opaque white limits, in centreblue pigment

6a2 R 8 mm irregular irregular hemispheric mucilaginous opaque actinomycetes withwhite aerian micelia

6a3 R 6 mm irregular whole umbonated dry opaqueactinomycetes withdowny white aerian

micelia6a4 S 3 mm round whole hemispheric mucilaginous opaque milky-white

6a

6a5 R 4 mm round whole hemispheric downy opaqueactinomycetes withdowny grey aerian

micelia6b1 R 1,9 cm round lobate flat dry opaque milky-white

6b6b2 S 3 mm round whole hemispheric mucilaginous opaque yellow-orange

6c1 R 5 cm filamentous irregular flat dry transparent actinomycetes withwhite aerian micelia

6c2 R 6 mm round irregular flat dry opaque white6c

6c3 S 2 mm round whole raised mucilaginous opaque orange

7a1 R 6 mm round lobate umbonated dry opaqueactinomycetes with

cream-coloredaerian micelia7a

7a2 R 4 cm round irregular hemispheric downy opaque actinomycetes withwhite aerian micelia

7b1 R 3 cm filamentous irregular flat dry transparent white

7b2 R 1,8 cm irregular serrated flat dry opaque milky-white7b

7b3 R 1,3 cm irregular serrated flat mucilaginous opaque milky-white

7Aa1 R 5 cm filamentous irregular flat dry opaque actinomycetes withwhite aerian micelia7Aa

7Aa2 S 4 mm round whole hemispheric mucilaginous opaque rosy

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7Aa3 R 7 mm round serrated flat dry opaque cream-colored with awhite ring

7y 7y1 R 2 mm irregular serrated flat mucilaginous opaque cream-colored

8 81 S 3 mm round whole raised mucilaginous opaque orange

91 R 8 mm round lobate umbonated mucilaginous opaque cream-colored

92 S 4 mm round whole raised mucilaginous opaque orange9

93 R 5 mm round whole umbonated downy opaqueactinomycetes withdowny white aerian

micelia10i 10i S 2 mm point-like whole flat dry transparent white

10ii1 S 3 mm round whole hemispheric dry opaque orange

10ii2 S 5 mm round whole hemispheric dry opaque yellow10ii

10ii3 R 6 mm round irregular hemispheric dry opaque actinomycetes withwhite aerian micelia

10iii 10iii S 4 mm round whole ridicat mucilaginous opaque yellow -orange

10iv 10iv S 1 mm point-like whole flat dry opaque beige

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We also aimed at describing the micro-morphology of isolated strains (Table III).From the 55 strains, 5 strains were represented morphologically by small Gram

negative unsporulated bacilli; one strain was represented by a Gram positive unsporulatedcoccobacillus; one strain – by Gram positive unsporulated bacillus; 6 strains – by smallGram positive bacilli, and 21 strains belong to Gram positive cocci. The microbial strainssubmitted to the microscopically examined can be classified into the following categories(Fig. 2), established by Tayler and Lochhead (quoted, Nimiţan Erica, 1997):

o Group I – short rods , Gram positive – 10,91 %o Group II – short rods, Gram negative – 9,09 %o Group III – short rods, Gram variable – 0%o Group IV – coccoide rods – 1,82 %o Group V – Gram positive or negative cocci – 38,18 %o Group VI – unsporulated long rods – 1,82 %o Group VII – sporulated rods – 38,18 %.

0

10

20

30

40

%

Morphological groups of bacteria

GROUP I

GROUP II

GROUP III

GROUP IV

GROUP V

GROUP VI

GROUP VII

Fig. 2 – Percentage representation of the main morphological groups of bacteria

from analyzed soil samples

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Table III - Micro-morphological aspect of isolated bacterial strains from soil samples

ANALYZEDSAMPLE

ISOLATEDSTRAINS

MORPHOLOGICAL TYPE

TINCTORIALAFFINITY

GROUPING MODE SPORULATIONCAPACITY

1 a1 bacillus Gram + diplo central andsubterminal spores,

undeforming1 a2 coccus Gram + diplo unsporulated1 a3 coccus Gram + tetrade unsporulated

1 a

1 a4 bacillus Gram + isolated spore mass1 b1 coccus Gram + tetrade unsporulated1 b2 bacillus Gram + coiled chains spore mass1 b3 bacillus Gram - isolated unsporulated

1 b

1 b4 coccus Gram + cubical pack (sarcina) unsporulated2 a1 bacillus Gram + diplo central spores,

undeforming2 a2 bacillus Gram - isolated unsporulated2 a3 coccus Gram + tetrade unsporulated

2 a

2 a4 bacillus Gram + isolated central spores,deforming

2 b1 coccus Gram + tetrade unsporulated2 b2 micelial hyphae

(actinomycet)Gram + - unsporulated

2 b2 b3 coccus Gram + cluster (staphylo-) unsporulated3 a1 bacillus Gram + chains spore mass3 a2 coccus Gram + cluster (staphylo-) unsporulated3 a3 a3 bacillus Gram + heaps unsporulated

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3 a4 coccus Gram + cluster (staphylo-) unsporulated3 b1 bacillus Gram + isolated spore mass3 b2 bacillus Gram + isolated unsporulated3 b3 bacillus Gram + diplo unsporulated3 b4 coccus Gram + cluster (staphylo-) unsporulated

3 b

3 b5 bacillus Gram + letters shape unsporulated4 a1 bacillus Gram - chains unsporulated4 a2 bacillus Gram + diplo unsporulated4 a3 micelial hyphae

(actinomycet)Gram + - unsporulated

4 a4 bacillus Gram + isolated spore mass

4 a

4 a5 bacillus Gram + chains central spores,undeforming

4 b1 coccus Gram + cluster (staphylo-) unsporulated4 b2 micelial hyphae

(actinomycet)Gram + - unsporulated4 b

4b3 bacillus Gram + coiled chains masă spori5 a1 Coccobacilli Gram + diplo unsporulated5 a5 a2 coccus Gram + isolated unsporulated5 b1 bacillus Gram + diplo central spores,

undeforming5 b2 coccus Gram + cluster (staphylo-) unsporulated5 b3 bacillus Gram - diplo unsporulated

5 b

5 b4 coccus Gram + tetrade unsporulated6 a1 bacillus Gram + isolated spore mass

6 a6 a2 micelial hyphae

(actinomycet)Gram + - unsporulated

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6 a3 micelial hyphae(actinomycet)

Gram + - unsporulated

6 a4 bacillus Gram + isolated spore mass6 a5 micelial hyphae

(actinomycet)Gram + - unsporulated

6 b1 coccus Gram + tetrade unsporulated6 b

6 b2 coccus Gram + sarcina unsporulated6 c1 micelial hyphae

(actinomycet)Gram + - unsporulated

6 c2 bacillus Gram + chains spore mass

6 c

6 c3 coccus Gram + cluster (staphylo-) unsporulated7 a1 bacillus Gram + heaps spore mass7 a

7 a2 micelial hyphae(actinomycet)

Gram + - unsporulated

7 b1 micelial hyphae(actinomycet)

Gram + - unsporulated

7 b2 bacillus Gram + isolated central spores,undeforming

7 b

7 b3 bacillus Gram + diplo spore mass7 Aa1 bacillus Gram + isolated spore mass

7 Aa2 micelial hyphae(actinomycet)

Gram + - unsporulated7 Aa

7 Aa3 bacillus Gram + isolated spore mass7 y 7y1 bacillus Gram + isolated spore mass

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8 81 bacillus Gram + isolated central and terminalspores, undeforming

91 coccus Gram + cluster (staphylo-) unsporulated

92 coccus Gram + isolated unsporulated9

93 micelial hyphae(actinomycet)

Gram + - unsporulated

10 i 10 i bacillus Gram - diplo unsporulated

10 ii1 coccus Gram + cluster (staphylo-) unsporulated

10 ii2 coccus Gram + tetrade unsporulated10 ii

10 ii3 micelial hyphae(actinomycet)

Gram + - unsporulated

10 iii 10 iii bacillus Gram + isolated spore mass

10 iv 10 iv bacillus Gram + isolated subterminal spores,undeforming

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As a whole, the results of the micro-morphological analyses for the bacterialstrains isolated in the soil indicated the following types: unsporulated Gram positive cocci,isolated or grouped as diplo-, tetrade, load or bunch; isolated unsporulated Gram negativebacilli; Gram positive bacilli, diplo-grouped, with central or subterminal non-deformingspores; isolated, completely sporulated Gram positive bacilli; completely sporulated Grampositive bacilli located in wreathed chains; completely sporulated Gram positive bacilligrouped in wreathed chains; isolated Gram positive bacilli, with central, deforming spores;completely sporulated Gram positive bacilli grouped in long chains; Gram positive bacilli,grouped in heaps; Gram positive bacilli grouped under letter-shapes; coccobacilli; longmycelial hyphae, thin, unsepted (Photos 2a, 2b, 2c, 2d).

From a quantitative point of view, we can notice a bacillus-type dominance in allthe soil samples analyzed. According to Bergey’s Manual of Systematic Bacteriology [5], weidentified the genera Bacillus, Clostridium (Gram positive bacilli) and Pseudomonas (Gramnegative bacilli), and, among the cocci, the genera Sarcina, Gafkia, Staphylococcus. Theresults obtained confirm the data in the specialty literature, according to which therepresentatives of the Bacillus genus can survive in the soil as spores. Non-sporogenousbacteria are represented by a series of Gram negative species, belonging especially to thePseudomonas genus.

Conclusions

We registered quantitative variations of the soil microbiota, according to the typesof soil analyzed and to the depth at which samples were collected, and we identified ahigher microbial load in the entianthroposoil types.

From the soil samples analyzed, 67 microorganism strains were isolated, from thebacteria (55 strains) and actinomycetes (12 strains) types. They were included in theCollection of the Laboratory of General Microbiology from Faculty of Biology, Iasi, andwill serve for further investigations.

Based on the macro- and micro-morphological characteristics studied, thebacterial strains that were isolated were classified into five morphological groups: smallGram positive bacilli, small Gram negative bacilli, coccobacilli, Gram positive cocci andsporulated bacilli, the main genera being Bacillus, Clostridium (Gram positive bacilli) andPseudomonas (Gram negative bacilli), and from cocci the genera Sarcina, Gafkia,Staphylococcus.

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BIBLIOGRAPHY

1. AKKERMANS, A.D.L.,VAN ELSAS, J.D., DE BRUIJIN, 1995 – Molecular microbial ecology manual,Kluywer Academic Publishers, Dordrecht The Netherlands: 75-77.

2. ALEXANDER, M., 1971 – Microbial ecology, J. Willey and Sons Inc., New York: 51-57.3. ANGLE, S., WEAVER, R.W., BOTZTOMLEY, P., BEZDICEK, D., SMITH, S., TABATABAI, A.,

WOLLUM, A., 1994 – Methods of soil analysis, part 2 – Microbiological and biochemical proprieties– Soil Science Society of America, Inc.: 92-121.

4. JOHNSON, T.R., CASE, C.L., 1998 – Laboratory experiments in microbiology – The Benjamin CummingsPublishing Company, Inc.: 124-131.

5. KRIEG, N.R., HOLT, J.G., 1984 - Bergey’s Manual of Systematic Bacteriology, vol. I, Williams & WilkinsCompany, Baltimore.

6. LIM, D.L., 1998 – Microbiology, McGraw-Hill Companies, Inc.: 418-436.7. MADIGAN, M.T., MARTINKO, J.M., PARKER, J., 2000 – Brock Biology of Microorganisms, Pretince Hall,

Inc. Upper Saddle River, New Jersey: 102-112.8. MĂZĂREANU, C., 1999 – Microbiologie generală, Editura Alma Mater, Bacău: 200-207.9. NIMIŢAN, ERICA, COMĂNESCU, ŞTEFAN, MARIN, ELENA, 1997 – Ecologia microorganismelor, Ed.

Cermi, Iaşi: 64-73.10. NORRELL, S.A., MESSLEY, K.E., 1997 – Microbiology laboratory manual, Principles and Applications –

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AKNOWLEDGEMENTS

The research had the financial support of the National University Research Council in the Grant no. 1174 on thetheme: 'Polluted soils mycoremediation in areas considered critical by ecological point of view '.

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a b

c dPhoto 1 – Aspect of microbial colonies isolated from different soil types:

a - 1 a (10-5);b - 6 a (10-5);c - 7 a (10-6);

d - 10 iii (10-5).

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da b

C dPhoto 2 – Microscopically view of the isolated bacterial strains (x 1000):

a - 1 a1; b - 2 b4; c - 5 b1;d - 6 c3.