210

ISSN 1064-2293, Eurasian Soil Science, 2008, Vol. 41, No. 2, pp. 210–214. © Pleiades Publishing, Ltd., 2008.Original Russian Text © G.M. Zenova, A.A. Gryadunova, A.I. Pozdnyakov, D.G. Zvyagintsev, 2008, published in Pochvovedenie, 2008, No. 2, pp. 235–240.

INTRODUCTION

The peat soils are products of special organic–accu-mulative soil-forming processes. Peat has an oxygendeficiency; biochemical processes and biologicalcycles of substances are inhibited there due to theexcessive atmospheric and ground-water moisteningand high water holding capacity [12].

The specificity of peat soils as a habitat—theappearance of anaerobic zones due to the seasonal fluc-tuations of the ground water table and the ability to holdgreat water reserves—suggests the presence of micro-organisms (including actinomycetes) that are able toexist under the oxygen deficiency in the soil air [10]. Inthe peat soils, practically all the groups of microorgan-isms decomposing peat are present [2, 4–6, 9]. The rep-resentatives of the

Streptomyces

genus were tradition-ally registered in peat soils. In the literature, there areonly some records about isolation of the representativesof so-called “rare” (rarely occurring) species of the

Thermoactinomyces

[1],

Nocardia

[20],

Agromyces

[18],

Micromonospora

,

Actinomadura

,

and

Streptospo-rangium

genera [10, 21]. In deep peat (depth of 3, 7 m),actinomycete mycelium was found in all the layers[3, 7].

The majority of the known soil actinomycetes areaerobic microorganisms with the oxidative type ofmetabolism and well-developed systems of transferringelectrons to oxygen. Nevertheless, under aerobic condi-tions, streptomycetes, micromonospores, and strep-toverticylls were isolated from flooded soils of rice pad-dies [9]. The dynamics of soil populations of anaerobicor microaerophilic organisms with rudimental myce-lium (

Actinomyces

and

Agromyces

) have been poorly

studied. Streptomycetes with unusual morphologiccharacteristics were isolated from a soddy-podzolic soilunder microanaerobic conditions [8].

The aim of this work is a comparative study of thedistribution and taxonomic identification of aerobic andmicroaerophilic actinomycetes in order to determinetheir position and role in the microbial complex of peatsoils.

OBJECTS AND METHODSA typical agropeat soil on forb–woody peat and a

typical peat soil on

Hypnum

–forb peat were studied inthe territory of the Dmitrov Division of the RussianResearch Institute of Agricultural Use of AmelioratingLands of Moscow Oblast (Table 1). The samples werecollected in the peat soils from depths of 0–20, 20–40,40–60, 60–80, and 80–100 cm. Five samples weretaken from each layer using the method of random sam-pling. Actinomycetes were isolated and counted on amedium with sodium propionate [11] with addition ofnystatin (50

μ

g/ml) or nalidixic acid (1.5

μ

g/ml) to sup-press the growth of fungi and nonmycelial bacteria,respectively.

The total number of actinomycete colonies wasdetermined. The differentiated count of their morpho-logical types was performed using an optical micro-scope (

×

400) [11]. Representatives of each type wereisolated as pure cultures and preliminarily identified(up to the genus level) according to the differentialtables of Bergey’s Manual of Determinative Bacteriol-ogy [14–17], as well as the morphological (fragmenta-tion and branching of mycelium, the presence and char-acter of the spore arrangement on air and/or substrate

SOILBIOLOGY

Aerobic and Microaerophilic Actinomycetes of Typical Agropeat and Peat Soils

G. M. Zenova, A. A. Gryadunova, A. I. Pozdnyakov, and D. G. Zvyagintsev

Faculty of Soil Science, Moscow State University, Leninskie gory, 119991 Russia

Received June 26, 2006

Abstract

—A high number (from tens of thousands to millions of CFU/g of soil) of actinomycetes and a highdiversity of genera were found in typical peat and agropeat soils. Agricultural use increases the number anddiversity of the actinomycete complexes of the peat soils. In the peat soils, the actinomycete complex is repre-sented by eight genera:

Streptomyces, Micromonospora, Streptosporangium, Actinomadura, Microbispora,Saccharopolyspora, Saccharomonospora

, and

Microtetraspora

. A considerable share of sporangial forms inthe actinomycete complex of the peat soils not characteristic of the zonal soils was revealed. The number ofactinomycetes that develop under aerobic conditions is smaller by 10–100 times than that of aerobic forms inthe peat soils. Among the soil actinomycetes of the genera

Streptomyces, Micromonospora, Streptosporangium,Actinomadura, Microbispora

, and

Microtetraspora

, the microaerophilic forms were found; among the

Saccha-ropolyspora

and

Saccharomonospora

, no microaerophilic representatives were revealed.

DOI:

10.1134/S1064229308020129

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AEROBIC AND MICROAEROPHILIC ACTINOMYCETES OF TYPICAL AGROPEAT 211

mycelium, the presence of sporangia, and the mobilityof spores) and chemotaxonomic characteristics (thepresence of the L- or mesoisomer of diaminopimelicacid (DAPa) and the differentiating sugars in hydrolyz-ates of intact cells) [19].

The total population of actinomycetes in the colony-forming units (CFU) per 1 g of substrate and the rela-tive abundance of representatives of the taxa foundwere determined in every soil sample. For each layer ofthe peat soils, the frequency of occurrence of represen-tatives of different actinomycete genera was deter-mined as the ratio between the number of sampleswhere the species of an actinomycete genus occurredand the total number of the samples analyzed.

For the identification of microaerophilic actino-mycetes in the peat soils, the soil suspensions wereinoculated under the agar layer and between two agarlayers in Petri dishes [13]. The colonies grown werecounted in the way described for aerobic actino-mycetes. All the colonies isolated were inoculatedagain into test tubes with a freshly autoclaved mediumby picking the whole agar thickness (7 cm). The abilityof the actinomycetes to grow on the surface of the agarand within the agar column was observed [13]. In thecultures growing within the agar column, the CO

2

emis-sion was measured upon the substitution of the air fornitrogen above the liquid nutrient medium in a penicil-lin flask. The air medium in the flask was substitutedusing the technique of nitrogen being blown throughthe flasks until the nitrogen content was 2% of the totalair volume. The hermetically closed flasks were incu-bated at

28°ë

. The

ëé

2

emission was measured usinga gas chromatograph with a detector for the heat con-ductivity (katharometer) in a column made of stainlesssteel (with an interior 2.0 mm in diameter and 3.2 m inlength) with Polisorb-1 adsorbent. Helium was used asthe gas carrier, which entered with a speed of30 ml/min. The temperature of the detector was

100°ë

;the current of the measuring elements was 148 mA; andthe temperatures of the thermostat and the injectionchamber were 30 and

40°ë

, respectively [13].

RESULTS

The method of surface plating used for the peat soilsallowed revealing a considerable population (from tensof thousands to a million CFU/g of soil) of actino-mycetes and identifying eight genera of mycelialprokaryotes in the actinomycete complex:

Streptomy-ces

,

Micromonospora

,

Streptosporangium

,

Actinoma-dura

,

Microbispora

,

Saccharopolyspora

,

Saccha-romonospora

,

and

Microtetraspora

.The actinomycete complexes of the peat soils are

comparable with those in chernozems with respect totheir number and diversity [10]. In zonal soddy-pod-zolic soil, the diversity of actinomycetes is limited to3

−

4 genera, and the population of actinomycetes doesnot exceed hundreds of thousands of CFU/g of soil[10]. The agricultural use of peat soils increases thenumber of actinomycetes and the diversity of the acti-nomycete complex.

The distribution of actinomycetes throughout theprofile of the peat soils was quite even with insignifi-cant fluctuations of their number in some peat layers. Ata depth of 60–80 cm, the actinomycete populationremained rather high (

10

4

–10

5

CFU/g of soil), whichagreed with the literature data [10].

In the agropeat and peat soils, the distribution of themycelial prokaryote complexes within their profileshad specific features. In the upper layers (0–20, 20–40 cm)of the agropeat soil, the number of all the actinomycetegenera isolated was greater by an order of magnitude ascompared to that in the same layers of the peat soil(Fig. 1). In the upper layers of the agropeat soil, the act-inomycete complexes were more diverse in terms of thegenera diversity than those in the same layers of thepeat soil. In the deeper layers of both peat soils, the den-sity of the actinomycete populations differed to a lesserdegree. In the agropeat soil, the diversity of the generadecreased little with the depth; in the peat soil, this trendwas practically not noted. In the deep layers of this soil, theactinomycete diversity was greater by 6–7 genera thanthat in the surface soil layers. The genera

Steptomyces,Micromonospora

, and

Streptosporangium

were perma-

Table 1.

Agrochemical properties of the peat soils

Soil and duration of its use Ash content, % C/N N

total

, % pH

water

N-N N-N water

mg/100 g

Typical agropeat soil on forb–woody peat, 85–90 years

37.08 12.9 2.45 6.4 1.32 2.34

Typical peat soil on Hypnum–forb peat, no data

15.4 12.9 1.3 7.5 0.69 3.31

O3– H4

+

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nently found in the peat soils (from tens to hundreds ofthousands of CFU/g of soil). In different layers of theagropeat soil, 4–5 rare actinomycete genera were iso-lated; in the peat soil, 3–4 were isolated.

The frequency of occurrence of the actinomycetesin space has revealed the genera that were most oftenisolated from the peat soils. In the agropeat soil, thegenera

Streptomyces, Micromonospora, Streptospo-rangium

, and

Actinomadura

(their frequency was80

−

100% in the upper soil layers) were often identified.In the peat soil, the frequency of the genus

Streptospo-rangium

was only 29% (Fig. 2), although this genus

composed a significant part of the actinomycete com-plex.

The number of actinomycetes developing undermicroaerobic conditions turned out to be smaller by1

−

2 times than that of aerobic forms (Fig. 3). The reg-ularities of the distribution of microaerophylic actino-mycetes within the soil profiles studied were the sameas for the aerobic forms. In the agropeat soil, their num-ber decreased insignificantly downward; in the peatsoil, it somewhat increased. The species diversity littlechanged with depth in the agropeat soil, but, in the peatsoil, it was higher in the deeper layers. The

Streptomy-ces, Micromonospora, Streptosporangium

, and

Actino-madura

genera were permanently found in all the lay-ers of the agropeat soil (the soil suspension was inocu-lated under the agar layer). These genera predominatedamong the microaerophylic forms of the actinomycetecomplex. Actinomycetes of the genera

Microbispora,Saccharopolyspora, Saccharomonospora

, and

Microtetraspora

were isolated only from the upperhorizons of the agropeat soil. The diversity ofmicroaerophylic forms in the peat soils was muchlower: only

Streptomyces

and

Micromonospora

werefound. Under microaerobic conditions, the genera

Act-inomadura, Streptosporangium

, and

Saccha-ropolyspora

were identified only in some layers of thepeat soil;

Microbispora, Microtetraspora

, and

Saccha-romonospora

were rarely recorded. Among themicroaerophylic actinomycetes, the representatives ofthermotolerant forms of the

Streptomyces

and

Micromonospora

genera were revealed.

Under microaerobic conditions, 79 actinomycetestrains were collected. Representatives of 17 strains didnot grow within the agar column. On the agar surface,the growth of exclusively aerobic actinomycetes was

Fig. 1.

The number of different actinomycete genera in the agropeat (a) and peat (b) soils. Designations here and further:

1—Strep-tomyces, 2—Micromonospora, 3

—

Streptosporangium

,

4—Actinomadura, 5—Microbispora, 6

—

Saccharopolyspora, 7—Saccha-romonospora

, and

8—Microtetraspora

; N—CFU/g of soil.

Fig. 2.

The frequency of occurrence of different actino-mycete genera (percentage of soil samples with the genusidentified of the total number of samples analyzed) in theagropeat (a) and peat (b) soils (0- to 20-cm layers).

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observed. Most of the cultures (62 strains) grew withinthe agar mass at a depth of 1 to 6 cm from the surfaceand did not spread over the agar surface. Only 5 cul-tures (4 actinomycetes) were able to grow in the agarcolumn up to the depth of 7 cm (Table 2). Some repre-sentatives of the

Streptomyces, Micromonospora,Streptosporangium

, and

Actinomadura

genera grew onthe agar surface, while the others were found within itsmass. The representatives of

Saccharopolyspora

and

Saccharomonospora

were recorded only on the agarsurface and those of

Microtetraspora

and

Micro-bispora

, only within its thickness.

The ability of actinomycetes to grow and emit CO

2

in the medium containing 90% nitrogen and 2% oxygen(pointing to the transition to microaerobic conditions)showed that among the 26 test cultures only 13 (repre-sentatives of the

Streptomyces, Micromonospora,Streptosporangium, Actinomadura, Microbispora

, and

Fig. 3.

The number of microaerophilic actinomycetes of different genera in the agropeat (a) and peat (b) soils.

Table 2.

The population of actinomycete strains of different genera on the agar surface and within the agar medium in thecolumns

GenusDepth of the agar column up to which the growth of the culture was recorded, cm

0 0–1 1–2 2–3 3–4 4–5 5–6 6–7 total

Streptomyces

9 8 11 11 8 4 1 4 56

Micromonospora

2 1 1 4

Streptosporangium

3 1 2 1 1 1 9

Actinomadura

1 2 1 4

Microbispora

1 1 1 3

Saccharopolyspora

1 1

Saccharomonospora

1 1

Microtetraspora

1 1

Total strains of dif-ferent genera

17 11 12 13 12 7 2 5 79

Note: Empty cells designate the absence of cultures.

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Microtetraspora

genera) were able to produce CO

2

.The most intense CO

2

emission (from 10

–9

mol CO2/mlon the 7 day of growth to 2–3 10–9 mol CO2/ml on the7 day) was registered in the cultures of Microbisporaand Microtetraspora strains.

CONCLUSIONS

In the actinomycete complex of the peat soils, thepresence of microaerophilic actinomycetes, whosepopulation was smaller than that of aerobic forms, wasrevealed. The representatives of the Streptomyces,Micromonospora, Streptosporangium, Actinomadura,Microbispora, and Microtetraspora genera turned outto be able to grow and evolve CO2 at an oxygen contentof 2% in the air.

The presence of aerobic and microaerophilic actino-mycetes in the peat soils appeared to be related to thepermanent changes in the water–air regime due to thefluctuations of the groundwater table both in the yearsand seasons with changing moisture. The groundwatertable in the peat soils is an important characteristic,which reflects their water regime and the regime ofmineral nutrition of plants.

ACKNOWLEDGMENTS

This work was supported by the Russian Foundationfor Basic Research (project no. 06-04-48165) and bythe Grant of the President of the Russian Federation forthe Support of Leading Scientific Schools (grantno. NSh-1518.2003.4).

REFERENCES

1. N. S. Agre, Taxonomy of Thermophilic Actinomycetes(NTsBI, Pushchino, 1986) [in Russian].

2. N. I. Gantimurova, “Microflora of Peat-Bog Soils,” inMicroflora of West-Siberian Soils (Nauka, Novosibirsk,1970), pp. 148–170 [in Russian].

3. A. V. Golovchenko, L. M. Polyanskaya, T. G. Dobro-vol’skaya, et al., “Spatial Distribution and Structure ofMicrobial Complexes in Bog–Forest Ecosystems,”Pochvovedenie, No. 10, 78–89 (1993).

4. A. V. Golovchenko, N. G. Dobrovol’skaya, and L. I. Ini-sheva, “Structure and Stocks of Microbial Biomass inOligotrophic Peat Bogs of the Southern Taiga in WesternSiberia,” Pochvovedenie, No. 12, 1468–1473 (2002)[Eur. Soil Sci. 35 (12), 1296–1301 (2002)].

5. A. V. Golovchenko, Yu. V. Sannikova, T. G. Dobro-vol’skaya, and D. G. Zvyagintsev, “Saprotrophic Bacte-

rial Complex of High-Moor Peats in Western Siberia,”Mikrobiologiya 74 (4), 545–551 (2005).

6. A. V. Golovchenko, T. A. Semenova, A. V. Polyakova,and L. I. Inisheva, “Structure of the Micromycete Com-plex of Oligotrophic Peat in the Southern-Taiga Subzoneof Western Siberia,” Mikrobiologiya 71 (5), 667–674(2002).

7. T. G. Dobrovol’skaya, L. M. Polyanskaya, A. V. Go-lovchenko, et al., “Microbial Pool in Peat Soils,” Poch-vovedenie, No. 7, 69–77 (1991).

8. D. I. Dyakova, G. M. Zenova, S. N. Sukhova, andL. V. Kalakutskii, “Morphological and PhysiologicalFeatures of Actinomycetes Isolated from Soil underMicroanaerobic Conditions,” Biol. Nauki, No. 8, 89–93(1985).

9. E. N. Zhdannikova, “Microbiological Characterizationof Peat-Bog Soils of Tomsk Oblast,” in WaterloggedForests and Bogs of Siberia (Akad. Nauk SSSR, Mos-cow, 1963), pp. 170–182 [in Russian].

10. D. G. Zvyagintsev and G. M. Zenova, Ecology of Actino-mycetes (GEOS, Moscow, 2001) [in Russian].

11. G. M. Zenova, Rare Genera of Soil Actinomycetes(Mosk. Gos. Univ., Moscow, 2000) [in Russian].

12. N. G. Kovalev, A. I. Pozdnyakov, D. A. Musekaev, andL. A. Pozdnyakova, Peat, Peat Soils, and Fertilizers(Moscow) [in Russian].

13. Methods of Soil Microbiology and Biochemistry (Mosk.Gos. Univ., Moscow, 1991) [in Russian].

14. Bergey’s Manual of Determinative Bacteriology, 7th ed.,Ed. by R. S. Breed, E. G. D. Murray, and N. R. Smith(Williams and Wilkins, Baltimore, 1957).

15. Bergey’s Manual of Determinative Bacteriology, 8th ed.,Ed. by J. A. Holt (Williams and Wilkins, Baltimore,1974).

16. Bergey’s Manual of Systematic Bacteriology, Ed. byS. T. Williams, M. Sharpe., and J. A. Holt (Williams andWilkins, Baltimore, 1989), Vol. 4.

17. Bergey’s Manual Determinative Bacteriology, 9 ed., Ed.by J. G. Holt, N. R. Krieg, H. A. Peter, et al., (Williamsand Wilkins, Baltimore, 1994).

18. L. T. Casida, “Observation of Microorganisms in Soiland Other Natural Habitats,” Appl. Microbiol. 18, 1065–1103 (1969).

19. T. Hasegawa, M. Takizawa, and S. Takida, “A RapidAnalysis for Chemical Grouping of Aerobic Actino-mycetes,” J. Gen. Appl. Microbiol. 29, 319–322 (1983).

20. S. Ishizawa and M. Araragi, “Actinomycetes Flora ofJapanese Soils: IV. Actinomycetes Flora of Peat Soils,”Soil Sci. Plant Nutr. 17, 68–74 (1970).

21. The Prokaryotes. A Handbook on the Biology of Bacte-ria. Ecophysiology, Isolation, Identification, Applica-tions, Ed. by A. Balows, H. G. Truper, M. Dworkin, et al.(Springer, Berlin, 1991).