Bryansk oblast, where the active oil pipeline Druzhba passes and the level of radioactive pollution after the. Chernobyl accident is rather high. Accidents along.
ISSN 10642293, Eurasian Soil Science, 2012, Vol. 45, No. 12, pp. 1169–1173. © Pleiades Publishing, Ltd., 2012. Original Russian Text © A.L. Stepanov, O.B. Tsvetnova, S.N. Panikov, 2012, published in Pochvovedenie, 2012, No. 12, pp. 1320–1324.
SOIL BIOLOGY
Changes in the Structure of the Microbial Community under the Influence of Oil and Radioactive Pollution A. L. Stepanov, O. B. Tsvetnova, and S. N. Panikov Faculty of Soil Science, Lomonosov Moscow State University, Moscow, 119991 Russia Received July 14, 2010
Abstract—Different variants of combined radioactive and oil pollution were simulated in a series of model experiments with soils contaminated with radioactive materials. In the soils with a 137Cs pollution density of 5395.5 kBq, the number of Aeromonas, Pseudomonas, and Rhodococcus representatives decreased, and the number of mycobacteria and fungi increased. The pollution of the soils with diesel fuel with up to 5 mL/100 g soil is accompanied by raising the number of hydrocarbonoxidizing microorganisms; it eliminates to some extent the negative influence of the radionuclides on the soil microbial community approaching the soil to its initial or background state. The high soil pollution with diesel fuel (30 mL/100 g) leads to a decrease in the population of the microbial community and the biological activity. In the growing of plants, the negative effect of the combined radioactive and oil pollution on the biological activity manifests itself less contrast ingly. DOI: 10.1134/S1064229312060129
INTRODUCTION Nowadays, oil and oil refinery products, heavy metals, and technogenic radionuclides are among of the most widespread pollutants of the environment. However, situations of combined pollution with ecotoxicants of different natures are rather frequent; therefore, investigations of the combined effects of these pollutants on living natural objects are very top ical. As shown earlier, a microbocenosis readily responds to the environment’s alteration, and changes in the structure of a microbial community and the microbiological characteristics are best suited for the diagnostics of the changes in soils [10]. The effect of oil and radioactive pollution each on the soil microbial community has been well studied [2–8, 11]. Never theless, studies devoted to the simultaneous influence of radioactive elements and oil pollution on the soil microbiota have not been conducted, although, in the world practice, there are some cases when oil pollution is superimposed on radioactive pollution. In the Rus sian Federation, a similar situation may be found in Bryansk oblast, where the active oil pipeline Druzhba passes and the level of radioactive pollution after the Chernobyl accident is rather high. Accidents along this oil pipeline have been observed many times. For instance, on July 31, 2006, at the 39th km of the Unecha–Mozyr pipeline (Surazhskii district, Bryansk oblast), a pipe break took place resulting in a spill of 100 t of oil over an area of approximately 1000 km2 (according to the data of the Federal Service for Supervision of Natural Reserves Usage) [16].
The aim of the studies is the assessment of the changes in the structure of the soil microbial commu nities under the combined pollution of soils with 137Cs and oil products taking into account the effect of the vegetation. OBJECTS AND METHODS The studies were conducted as model experiments simulating different variants of the combined pollu tion of soils with 137Cs and oil products in the presence and absence of vegetation. The samples for the model experiment were collected in forests of the Bryansk Poles’e with the same phytocenotic (60yearold pine forests) and soil characteristics (sandy podzols on gla ciofluvial deposits) but with different levels of radioac tive pollution [1, 4]. Plot 1 (background) was chosen in the Starodubskii Forestry (Starodubskii district, Bryansk oblast) in the territory accepted as the background. At the moment of the investigations, the radioac tive pollution density of the soils with 137Cs averaged 3.33 mBq/m2 [14]. Plot 2 was located at the Krasnogorskii Forestry in the territory exposed to the strongest radioactive pol lution due to the accident at the Chernobyl atomic electric power station. Here, the mean density of the radioactive 137Cs pollution of the soil was 5395.5 mBq/m2 [14]. For the performance of the model experiment on studying the structure of the microbial community under the combined oil and radioactive pollution on
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microorganisms. Three replicates and the standard methodology were applied [9].
Scheme of the Experiment Plot 1 (background)
Simulation of oil pollution 60% of the TWC
Sample type Control
2 (contaminated with 60% of the TWC radioactive materials) 1 (background)
60% of the TWC + Zone of stress 5 mL DF/100 g soil
2 (contaminated with 60% of the TWC + radioactive materials) 5 mL DF/100 g soil 1 (background)
60% of the TWC + Zone of mor 30 mL DF/100 g soil tality
2 (contaminated with 60% of the TWC + radioactive materials) 30 mL DF/100 g soil
the test plots, mixed soil samples were taken from the humusaccumulative horizon (0 to 5cm layer) dis playing the highest density of radioactive pollution. The samples were dried at room temperature, care fully mixed, and the plant residues and admixtures were removed from them. Three samples (100 g each) were taken from the prepared soil. Each sample was put into a glass flask (500 mL), moistened up to 60% of the total water capacity (TWC), and simulated different variations of oil and radioactive pollution (table). Diesel fuel (DF) was chosen as the soil pollutant; it is the oil product whose composition is the closest to that of crude oil. A similar experiment was performed for the assess ment of the influence of the vegetation on the struc ture of the microbial community. For this purpose, 5dayold oat (Avena sativa) seedlings were planted on the soil of the samples taken. The method of gas chromatography–mass spec trometry was used for analyzing the microbial com munity [12, 15]. The essence of the analysis consists in the direct isolation of the higher fatty acids, aldehydes, and sterines and their separation using a chromato graph in a capillary column of high resolution and analyzing using a mass spectrometer under dynamic conditions. The final result was the determination of the concentration of a microbial marker and the sub sequent reconstruction of the composition and struc ture of the microbial community. The data of the cali bration according to deuterated tridecanoic acid and pure cultures of microorganisms were used for the cal culation [14]. The method of limited dilution of the culture was used for the determination of the number of soil
RESULTS AND DISCUSSION The analysis of the structure of the microbial com munity in the soils studied showed that, under the radioactive soil pollution, the microbial complex was transformed as compared to that in the background soil. The microbial diversity changed due to a decrease in the number of representatives of the Aeromonas, Pseudomonas, Methylococcus, and Clostridium genera and an increase in the number of mycobacteria, which are more characteristic of unfavorable conditions (Fig. 1). It is worth noting that the biomass of the fungi signifi cantly increased, which may be explained by their high resistance (especially of the darkcolored forms) to radioactive pollution [3]. Under the radioactive pollution, the addition of diesel fuel to the soil in the maximal dose (30 mL/100 g soil) was accompanied by a considerable growth of the number of microorganisms that decompose hydrocar bons (Rhodococcus and Pseudomonas species) (Fig. 1). In our opinion, the reduction of the number of fungi under the radioactive pollution is determined by the changes in the physical properties of the soils; the deterioration of the aeration; and, as a result, the reduction of the share of mycelial organisms. The absence of active oil destroyers among the fungi is also explained by this circumstance [8]. In the experimental variant with a dose of 5 mL/100 g, the number of Rhodococcus, Pseudomonas, and Aero monas species increased. The representatives of pre cisely these genera were depressed under the radioac tive pollution without the diesel fuel added. One can conclude that the application of diesel fuel in a dose up to 5 mL/100 g eliminates to some degree the influence of the radionuclides on the soil microbial community, thus approaching the soil to its initial or background state (Fig. 2). The growing of oats on the contaminated soils was accompanied by considerable changes in the structure of the soil microbial community. In the control, as compared with the model experiment without plants, the number of Caulobacter, Nocardia, Propionibacte rum, and Streptomyces species and some others decreased. The application of diesel fuel in low doses drastically increased the number of mycobacteria and decreased the number of propionic bacteria, Strepto myces, and Phodococcus. In the variant with the combined (radionuclides and diesel fuel) pollution of the soils in the absence of plants, the changes in the microbial community were the greatest and accompanied by an increase in the number of mycobacteria and sporeforming gram positive bacteria and the reduction of the share of EURASIAN SOIL SCIENCE
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Rhodococcus terrae
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Pseudomonas putida Eubacterium lentum
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Pseudomonas fluorescens Micrococcus/Arthrobacter
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Xanthomonas sp. Butyrivibrio 7S143
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Pseudonocardia sp. Streptomyces–Nocardiopsis
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Methylococcus/Clostridium sp. Nocardia sp. Aeromonas hydrophila Sphingomonas capsulata Acetobacterium sp. Rhodococcus equi Cytophaga sp. Sphingobacterium spiritovorum Corynebacterium sp. Bacteroides ruminicola Sphingomonas adgesiva Actinomadura roseola Clostridium difficile WolinellaAcholeplasmaRoseomonasBurkholderia Bifidobacterium sp. Protozoa Mycobacteria, total Bacillus subtilis Caulobacter AcetobacterRhodobacter group C. perfringens Eucariotes Pseudomonas freudenreichii Fungi 18 : 2, µg/g
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12 14 cell/g × 106
Fig. 1. The composition of the soil microbial community in the test variants without plants: 1—control (background plot); 2— radioactive pollution (5395.5 kBq/m2); 3—control with oil pollution (+5 mL/100 g of DF); 4—radioactive and oil pollution (+5 mL/100 g of DF); 5—radioactive and oil pollution (+30 mL/100 g of DF).
Nocardia, Acetobacter, Streptomyces and some other bacteria. The assessment of the total number of microorgan isms by the method of limited dilution of the culture showed that, in the background soil under oats, the total number of microorganisms reached 108 cell/g EURASIAN SOIL SCIENCE
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soil, and the radiation effect was not accompanied by a significant decrease in the number of microbial cells. In the diesel fuel pollution of the soil, the total number of microorganisms fell to 106 cell/g due to the reduc tion of the nonprofiled part of the microbial commu nity, for instance, of Streptomyces and some others.
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P. putida Peptostreptococcus Eubacterium lentum
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Pseudomonas fluorescens Propionibacterium+
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Micrococcus/Arthrobacter Xanthomonas sp. B. hypermegas/Selenomonas
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Butyrivibrio 7S143 Pseudonocardia Streptomyces Methylococcus/Clostridium sp. Nocardia carnea Aeromonas hydrophila Sphingomonas capsulata Eubacterium Acetobacterium sp. Rhodococcus equi Cytophaga Corynebacterium sp. Bacteroides ruminicola Sphingomonas adgesiva Actinomadura roseola Nocardiopsis WolinellaAcholeplasmaRoseomonasBurkholderia Bifidobacterium Protozoa Mycobacteria, total Bacillus subtilis Caulobacter AcetobacterRhodobacter group C. perfringens Eucariotes Pseudomonas freudenreichii Fungi 18 : 2, µg/g
0
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10 12 cell/g × 106
Fig. 2. The composition of the soil microbial community in the test variants with the participation of plants: 1—control (back ground plot, without plants); 2—control (background plot); 3—oil pollution (+5 mL/100 g of DF); 4—radioactive and oil pol lution (+30 mL/100 g of DF).
The increase in the diesel fuel concentration in the soil to 30 mL/100 g in the background of the radioactive pollution led to a decrease in the total number of microorganisms down 104 cell/g soil.
CONCLUSIONS Under radioactive soil pollution, the structure of the microbial community is reconstructed. In increasing the density of the soil 137Cs pollution to 5395.5 kBq/m2, the EURASIAN SOIL SCIENCE
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total number of Rhodococcus, Pseudomonas, and Aer omonas species sensitive to radioactive pollution decreases, and that of mycobacteria and fungi increases. The weak diesel fuel pollution (up to 5 mL/100 g) is accompanied by raising the number of hydrocarbon oxidizing microorganisms and, as the previous data showed, by intensifying the respiration, nitrogenfix ing activity, and denitrification [13]. Thus, the application of diesel fuel in low concen trations (probably, of other natural polymers available for microbial decomposition) somewhat eliminates the negative effect of radionuclides on the soil micro bial community promoting the approaching of the soil to the initial or background state. The high diesel fuel pollution (30 mL/100 g) of the soil leads to a reduction in the number of microorganisms and all the charac teristics of the biological activity studied [14]. The negative effects of the combined pollution with radionuclides and diesel fuel on the soil microbial community in the growing of plants manifest them selves to a lesser degree, probably, due to the positive influence of the root exudates on the soil microbial community.
1. Atlas of Cesium Contamination of Europe after the Cher nobyl Disaster (EK/IGKE, Rosgidromet; Mincherno byl’ (Ukraine); and Belgidromet, 1998). 2. Degradation and Conservation of Soils, Ed. by G. V. Dobro vol’skii (Izd. Mosk. Gos. Univ., Moscow, 2002) [in Rus sian]. 3. T. V. Denisova and K. Sh. Kazeev, Tolerance of Soil Microorganisms and the Biological Activity of Cher nozems toward Ionizing Radiation (Rostizdat, Rostov onDon, 2007) [in Russian]. 4. A. L. Efremov, “Biological Activity and Dynamics of Application Material Decomposition in Soils of Forest Biogeocenoses Polluted by Radionuclides,” Eur. Soil Sci. 32 (10), 1140–1146 (1999). 5. A. L. Efremov, “Dynamics of Soil Microflora and Microbial Metabolites under Conditions of the Radio
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