STEPANOV Aleksey (1), HATTORI Tsutomu (2), MANUCHAROVA Natalia (3), POLANSKAYA Lubov (4)

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1 Scientific registration no: 716 Symposium no: 9 Presentation: poster Microbial transformation of carbon and nitrogen compounds within soil aggregates Transformation microbienne des composés carbones et azotes dans les agrégats du sol STEPANOV Aleksey (1), HATTORI Tsutomu (2), MANUCHAROVA Natalia (3), POLANSKAYA Lubov (4) (1,3,4) Moscow State University, Soil Science Department, Moscow, Russia (2) Institute of Genetic Ecology, Tohoku University, Katahira, Aoba, 980 Sendai, Japan INTRODUCTION Microbial transformation of carbon and nitrogen compounds in the soils is carried out by microorganisms within soil aggregates. Localisation of microorganisms inside soil aggregates determines whether the type of metabolism carried out under aerobic or anaerobic conditions. Anaerobic zones within soil aggregates have been invoked to explain development of denitrifying process in aerated soils. But there is not experimental data determined the minimal soil aggregates diameter which limits of strictly anaerobic processes such as methane production and development of methanogenic archeabacteria in soil aggregates in the atmosphere with ambient oxygen level. The aim of present work is study distribution of microbial cells and their activity (production of carbon dioxide, nitrous oxide and methane) inside soil aggregates in aerated conditions. The results provides some explanation on the behaviour of aerobic and anaerobic microorganisms within soil aggregates. MATERIALS and METHODS Soil samples of chernozem were collected in the region of Voronez (Russia). Basic characteristics of the soil used in the experiments were as follow: organic matter (humus, %) - 11; ph in water - 7.4; moisture (%) - 40; temperature ( o C) The soil samples were sieved and spherical aggregates ranging from 2.0 to 12.0 mm in diameter were selected. Aggregates were handled as carefully as possible to minimize any disturbance to their surface. One gram samples of the five different aggregate sizes were placed in 15 ml germetic tubes, replicates 3 times. Glucose solution (0.2 ml) was added to each tube to obtain a final concentration of glucose 2.5 mg g -1 soil. The tubes were then preincubated for 30 days. Subsequently, the tubes were opened for air circulation, and glucose solution was again added to a similar concentration as described above. Total soil moisture was 0.4 ml g -1 soil. Decrease of oxygen content inside the tubes due to sampling of gas was compensated by injection of equal amounts of air. Denitrification rate was measured by acetylene inhibition of N 2 O reduction in 1

2 presence of calium nitrate (KNO mg g -1 ). Assessment of CH 4 production rates was measured in another series of tubes without acetylene. The gases (CO 2 and N 2 O) were measured by TDC (thermal conductivity detector) and separated on a Polysorb-1 column (3.0 m by o.3 o.d.) at 30 o C using helium as a carrier at a flow rate of 25 ml min -1. CH 4 was measured by FID (flame ionization detector) on a Spherosil column (3.5 m by 0.4 cm o.d.) at 40 o C using argon as a carrier at a flow of 30 ml min -1. Oxygen was detected by gas analyzer Gazochrom-401. Production rates of the gases were expressed on the basis of gram dry weight soil and were corrected for withdrawn gases. Samples (0.2 g) from central and surface parts of aggregates were placed into flasks with 50 ml of sterile water. The suspension was treated in an UZDN-1 ultrasonic desintegrator (0.44 A, 22 khz, 2 min). Total number of microorganisms was determined by luminescence microscopy. For quantifying the number of bacteria and the length of actinomyces mycelium the specimens were colored by a solution of acridine orange (1:10000 during 2-3 min). RESULTS Distribution of the fungi and bacteria inside and on the surface of soil aggregates showed that the length of fungi mycelium and average diameter of fungal hyphae and spores were greate on peripherical side of aggregates than in their central part. The average diameter of fungal spores and hyphae were 6.8 mkm and 4.6 mkm on the outside of aggregates and they comprised 4.2 mkm and 3.7 mkm in the center. Bacteria quantity and actinomycetes micelium increased within soil aggregates and was 7.8 x 10 7 and 5.4 x 10 6, respectively. Peculiar distribution of microorganisms and their activity in transformation of organic matter in central and on the surface of soil aggregates resulted formation of anaerobic zones within them. Total carbon dioxide production during 5 hours after glucose addition from small aggregates (diameter 2.0 mm) was 13.9 mg CO 2 g -1, wheras from large aggregates ( mm in diameter) was 8.0 and 7.9 mg CO 2 g -1, respectively. CO 2 production rate increased lineary with the surface area of soil aggregates caused fast formation of anaerobic microzones within soil aggregates of 2.0 mm diameter due to high rate of oxygen consumption in their periferical part mostly by fungal micelium. The study of anaerobic processes inside of soil aggregates of different sizes in oxygen atmosphere showed accumulation of nitrous oxide in gase phase due to development of denitrifying bacteria. Methane production was observed in all tests even under the presence of O 2 in the atmosphere. Oxygen concentration during the experiments was kept at a level of 20-17%. In general, production of CH 4 during a five days period from aggregates with a diameter 2.0, 6.0 and 12.0 mm was 3.1; 3.4 and 4.0 ng CH 4 g -1, respectively. Obtained results indicates that anaerobic processes such as denitrification and methane production can occur within soil aggregates of mm diameter and indicates that strictly anaerobic methanogenic archeabacteria can survive and become active inside small soil aggregates of 2.0 mm diameter even in an atmosphere with ambient oxygen level. DISCUSSION The present study showed that strictly anaerobic processes such as methane production occur within small soil aggregates of 2.0 mm in diameter even in the presence of atmospheric oxygen. Such results is not expected from the original model of Greenwood (1961). He suggested that an 2

3 anaerobic zone occurs in spherical soil aggregates as a function of aerobic respiration rate and the oxygen partial pressure surrounding them, assuming uniform distribution of microorganisms. The model proposed the presence of anaerobic center. Greenwood and Goodman (1967) proposed that the smallest aggregate containing an anaerobic center would have a radius of mm (minimal distance from surface to anaerobic zone within soil aggregate). However, oxygen distribution inside an individual soil aggregate showed that O 2 gradient was somewhat irregular, suggesting nonuniformly distributed sites of oxygen consumption (Sexstone et al., 1985). The contour map of O 2 distribution within soil aggregates showed that anaerobic zones were often asymmetric and did note always occur at the aggregate center. This suggests that the activity of oxygen cosumption within soil aggregates are not uniformly distributed, presumably due to a nonuniform distribution of soil microorganisms inside of the aggregates. Smith (1980) showed that a change in the diffusion coefficient of O 2 within soil aggregates had a greater effect on the extent of anaerobiosis than a comparable change in mean aggregates size. Sexstone et al. (1985) used respiration rate and intra-aggregate oxygen diffusion coefficient to calculate the anaerobic radius. However, some aggregates with anaerobic microsites measured by oxygen microelectrode (r mm) had no calculated anaerobic radius and the denitrification rate did not correspond to aggregate size or to the calculated anaerobic radius. This would suggest that anaerobic processes, such as methane production, can be carried out in microsites. Hattori (1988, 1993) proposed that in soil aggregates bacteria, especially gram negative, are distributed heterogenously; most of them are found in capillary pores which may have a radius less than 250 µm as cofirmed by Kilbertus (1980). Edwards and Bremner (1967) distinguished two categories of aggregate structure; microaggregates which are less than about 250 µm in diameter and are water-stable and macroaggregates which are composed of microaggregates that are easily destroyed by releasing microaggregates. Based upon their concept of the aggregate structure, we can consider that bacteria exist mostly within microaggregates, and consequently, the anaerobic sites of methane production are formed within such microaggregates. REFERENCES 1. Edwards A.P. and Bremner J.M Microaggregates in soil. J.Soil Sci. 18: Greenwood D.J The effect of oxygen concentration on the decomposition of organic materials in soil. Plant Soil 14: Greenwood D.J. and Goodman D Direct measurement of the distribution of oxygen in soil aggregates and in columns of fine soil crumbs. J.Soil Sci. 18: Hattori T Soil aggregates as microhabitats of microorganisms. Rep.Inst.Agr.Res. Tohoku University 37: Hattori T Protozoa in soil microhabitats. ISK serie N o.3:62, Inst.of Genetic Ecology, Tohoku University, Sendai, Japan. 6. Kilbertus G Etude des microhabitats contenus dans les agregats du sol. Leur relation avec la biomasse microbienne et la taille des procaryotes present. Rev.Ecol.Biol.Sol 17: Sexstone A.J., Revsbech N.P., Parkin T.N. and Tiedje J.M Direct measurement of oxygen profiles and denitrification rate in soil aggregates. Soil Sci.Soc.Am.J. 49: Smith K.A A model of the extent of anaerobic zones in aggregated soils, and its potential application to estimates of denitrification. J.Soil Sci. 31: Key words: aggregates, carbon dioxide, methane, nitrous oxide, production. Mots cles: agrégats, gaz carbonique, méthane, oxydes d azote, production 3

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