paleosols buried under kurgans. It was found that the total microbial biomass (including microbial cells cor- responding to different stages of the life cycle) in dif-.
ISSN 1064-2293, Eurasian Soil Science, 2008, Vol. 41, No. 13, pp. 1439–1447. © Pleiades Publishing, Ltd., 2008.
SOIL BIOLOGY
Age and Activation of Microbial Communities in Soils under Burial Mounds and in Recent Surface Soils of Steppe Zone T. S. Demkinaa, T. E. Khomutovaa, N. N. Kashirskayaa, E. V. Demkinab, I. V. Stretovicha, G. I. El-Registanb, and V. A. Demkina a
Institute of Physicochemical and Biological Problems of Soil Science, Russian Academy of Sciences, ul. Institutskaya 2, Pushchino, Moscow oblast, 142290 Russia b Vinogradskii Institute of Microbiology, Russian Academy of Sciences, pr. 60-letiya Oktyabrya 7/2, Moscow, 117811 Russia Received March 21, 2008
Abstract—Chestnut paleosols buried under steppe kurgans about 4800, 4000, and 2000 years ago and their background analogues were studied in the dry steppe zone on the Volga–Don interfluve. Morphological, chemical, microbiological, biochemical, and radiocarbon studies were performed. Paleoclimatic conditions in the region were reconstructed on the basis of paleosol data. The ages of microbial fractions isolated from the buried and surface soils were determined using the method of 14C atomic mass-spectrometry. It reached 2100 years in the A1 horizon of the buried paleosol, which corresponded to the archaeological age of the kurgan (1st century AD). The ages of microbial biomass isolated from the B2 horizons of the buried paleosol and the background surface soil comprised 3680 ± 35 and 3300 ± 30 years, respectively. The obtained data confirmed our assumption about preservation of microorganisms of the past epochs in the paleosols buried under archaeological monuments. It is ensured by various mechanisms of adaptation of soil microbial communities to unfavorable environmental conditions (anabiosis, transformation of bacteria into nanoforms, etc.). The possibility to stimulate germination of the ancient dormant microbial pool isolated from the buried paleosols by 2–3 orders of magnitude with the use of β-indolyl-3-acetic acid as a signal substance was demonstrated. DOI: 10.1134/S1064229308130139
INTRODUCTION Problems of the development and functioning of soil microorganisms in the course of soil evolution during the historical time remain poorly studied by soil microbiology. Investigations into the state of microbial communities in paleosols buried under archaeological monuments (steppe kurgans) of different ages represent one of the ways to solve these problems. Microbiological investigations performed in the recent decade have shown that the microbial communities of paleosols buried under steppe kurgans preserve some of their properties from the moment of soil burying (kurgan construction) up to the present time. Special studies of these properties make it possible to restore the conditions of soil functioning in the given historical epochs [9–12, 15, 23]. The analysis of distribution of microorganisms in the buried paleosols and in the kurgans demonstrated that the number of microorganisms in the A1 horizon of buried paleosols is on order of magnitude higher than that in the body of kurgans [13]. This fact also attests to the preservation of soil microbial community since the moment of kurgan construction. Thus, indirect data suggest that the age of microbial communities in the buried soils should be equal to the age of the soil burying (kurgan construction). However, there are no direct determinations of the age of microbial cells in the paleosols buried under kurgans. It was found that the total microbial biomass (including microbial cells cor-
responding to different stages of the life cycle) in different horizons of the paleosols under kurgans varies from 624 to 1746 µg C/g soil, whereas the mass of microorganisms sensitive to the application of glucose (i.e., microorganisms in the active state) is much lower (0.4–94 µg C/g soil) [28]. Hence, a larger part of the microbial community in the buried paleosols is in a dormant state. In this context, the capacity of “ancient' microorganisms from paleosols buried under archaeological monuments to be activated should be investigated. Our work was aimed at determining the age of microbial communities in paleosols buried under kurgans; their potential for activation was also analyzed. OBJECTS AND METHODS The area of our studies is found on the Volga–Don interfluve. The climate is moderately continental; the mean annual precipitation is 350 mm, and the mean annual air temperature is +8°C; the hydrothermal coefficient (the precipitation-to-evaporation ratio) equals 0.5. The snow cover depth averages 10 cm. The depth of soil freezing reaches 70–100 cm. Gently undulating interfluves and smooth slopes of river valleys and ravines predominate in the relief. Natural vegetation is represented by the wormwood–fescue association. This
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area belongs to the zone of dry steppes with chestnut soils (often, in complexes with solonetzes). The Aksai key plot in the northern part of the Ergeni Upland is found 100 km to the southwest of Volgograd (2.5 km to the southeast of the village of Aksai in Oktyabr’skii district). Background surface soils and paleosols buried under kurgan Aksai-3 were studied. The kurgan is found on the flat top of local watershed at 100–110 m a.s.l. The soils are developed from calcareous and saline loesslike loams; the groundwater table is at a depth of 30 m. Archaeological excavations were performed by the archaeological expedition of Volgograd State University under the supervision of A.N. D’yachenko. The Avilovskii key plot in the southern part of the Privolzhskaya Upland is found 80 km to the north of Volgograd (1 km to the west of the village of Avilovskii in Ilovlinskii district). The burial mound is situated on the high first terrace on the right bank of the Ilovlya River (left tributary of the Don River) at 50–60 m a.s.l. The terrace surface is dissected by numerous ravines ensuring good drainage; the groundwater table is found at a depth of more than 10 m. The soils develop from thick (>4–5 m) loesslike loams underlain by the alluvial fine-grained sand. Wormwood–grassy and wormwood–chrysanthemum–grassy associations predominate in the plant cover. Chestnut soils predominate on the terrace. Archaeological excavations were performed by the archaeological expedition of Volgograd State University under the supervision of a candidate of historical sciences I.V. Sergatskov. Samples for the microbiological analyses were taken from the soil genetic horizons with necessary sterility measures. Before the analyses, the samples were sieved through a 3-mm sieve mesh 3 mm to remove roots; averaged samples were analyzed. The age of microbial community was determined in the microbial fraction by the method of 14C atomic mass-spectrometry in the Radiocarbon Laboratory of Poznan Science and Technology Park in Poland. The microbial fraction was isolated from the soil using the method of cell extraction [28]. The method included the following procedure: the ultrasonic treatment of soil suspension (two times for 30 s with a pause of 30 s upon the suspension cooling) on an UZG13-01/22 and the separation of supernatant liquid with microbial cells from the soil via centrifuging at 2000 rpm for 30 min. After three successive treatments, supernatant solutions were joint together, and the microbial fraction was settled down via centrifuging at 5000 rpm for 90 min. Then, it was washed with sodium pyrophosphate to remove possible humus residues, lyophilized, and weighed; the organic carbon content in the microbial cells was determined. To activate germination of bacteria, the soil water suspensions with added β-indole 3-acetic acid (IAA) (in concentration of 10–4 M) and without it (control samples) were preincubated for 40 min on an agitator.
Then, inoculation of the suspensions into rich nutrient medium (RM: 3 g of dry agar, 3 g of peptone, 1 g of tryptone, 1 g of yeast extract, 1 g of glucose, and 20 g of agar-agar per 1000 ml of tap water) was performed [2]. The total number of microbial cells in the soil was determined by the method of direct counting under a luminescent microscope using a fluorescent dye DAPI (4, 6-diamino-2-phenilindol) that stains both living and dead cells. The total microbial biomass (C-MB) was calculated from data on the content of microbial fraction with a correction for the completeness of the extraction procedure; the latter was judged from the numbers of soil microorganisms forming their colonies on soil agar [22]. The carbon of active microorganisms (C-SIR) was estimated from the rate of substrate-induced respiration [1] using the conversion factor of 40.04 [25]. The soil microbial communities were characterized by the numbers of organisms growing on the rich nutrient medium (RM), soil agar (SA), and nitrite agar (NA) [21]. The ecological-trophic structure of microbial communities (the ratios of microorganisms grown on soil agar, nitrate agar, and rich medium (SA : NA : RM)) were determined; the oligotrophy index SA/RM × 100 [18] was also calculated. Mycelium of microscopic fungi was determined with the help of Hansen’s method of membrane filters modified by Demkina and Mirchink [14] to differentiate between the light- and dark-colored hyphae. All the parameters were determined in triplicate. The statistical treatment of experimental data was performed using the COHORT program [26] with application of COSTAT and ANOVA procedures. The criterion of the least significant difference (LSD) at the 5% confidence level was used to judge the difference between the experimental variants. RESULTS AND DISCUSSION Morphological and Chemical Properties of Buried Paleosols and Background Surface Soils The Aksai plot. Buried paleosol (pit D-681) was studied under kurgan no. 3. According to archaeological evidence, this kurgan was created in the first century AD (about 2000 years ago, during the Middle Sarmatian Culture). The modern height of the kurgan in its central part is 1.5 m, and the diameter reaches 30 m. The kurgan body is composed of the material from the A1 and B1 horizons of the ancient soil, and this fact determines the absence of soluble salts, gypsum, and carbonates of diagenetic origin in the upper part of the buried paleosol. The paleosol can be classified as a solonetzic and deeply solonchakous chestnut soil. Its characteristic features (Tables 1, 2) are as follows: a reddish brown color and a small-prismatic structure of the solonetzic B1 horizon with a considerable amount EURASIAN SOIL SCIENCE
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Table 1. Morphological and chemical characteristics of the buried paleosol and background surface soil at the Aksai site Solonetzic deeply solonchakous Modern solonetzic deeply solonchestnut paleosol under the kurgan, chakous chestnut soil (pit D-678) 1st century AD (pit D-681)
Parameter Thickness of the humus layer (A1 + B1), cm Color, A1 horizon Color, B1 horizon Structure, B1 horizon Concentrations of manganese oxides in the B1 horizon Depth of effervescence, cm Depth of accumulation of carbonates, cm Mean weighted CaCO3 content (0–50 cm), % Depth of accumulation of soluble salts, cm Mean weighted content of soluble salts (0–170 cm), % Depth of gypsum accumulation, cm Mean weighted gypsum content (0–170 cm), %
32 Light gray Reddish brown Relatively weak small prismatic Abundant films and nodules 32 35 3.1 110 0.64 110 1.15
29 Gray Brown Coarse prismatic Absent 20 25 7.3 90 0.81 90 2.63
Table 2. Chemical properties of the buried paleosol and background surface soil at the Aksai site Horizon; depth, cm
A(kurgan), 0–30 C(kurgan), 30–143 A1 143–153 B1 153–175 B2ca 175–187 BCca 187–227 C 227–255 Cs,y 255–298 Cs 298–310 A1 0–18 B1 18–29 B2ca 29–50 BCca 50–94 Cs,y 94–140 Cs,y 140–170
Humus, %
pH H2 O
Total salts, %
CEC, meq/100 g
CaCO3, %
Gypsum, %
Contents of particles, %
