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Scientific registration no : 833 Symposium no :26 Presentation : poster Nitrous oxide production by soil microscopic fungi Production d'oxyde nitreux par les champignons microscopiques du sol KURAKOV Alexander , PAKCHNENKO Oksana, KOSTINA Natalia, UMAROV Marat Moscow Lomonosov State University, Soil Science Faculty, Soil Biology Department and International Biotechnology Center, 119899, Moscow, Russia Many species of soil microscopic fungi of classes Deuteromycetes and Ascomycetes were capable to produce nitrous oxide at the conditions of reduced pressure of oxygen and the presence of nitrite and nitrite with peptone in media. Few strains of Fusarium oxysporum and F. solani released of nitrous oxide on the medium with nitrate. Most active producers of nitrous oxide belonged to Fusarium oxysporum, F. solani, Chaetomium globosum, Neosartorya fisheri, Trichoderma, Penicillium expansum, Talaromyces and some others species. Activities of nitrous oxide production by microscopic fungi was on several orders lower than denitrifying bacteria. The production of nitrous oxide by fungi was 2-240 mM N2O for 7 days. The emission of nitrous oxide was detected from sterile soil inoculated by fungal producers of N2O and their incubation at anaerobic and microaerobic conditions. It is well-known that bacteria carried out processes of denitrification, nitrification, dissimilatory nitrate reduction to ammonium responsible for nitrous oxide production. There were reports that some species of microscopic fungi release nitrous oxide on liquid media (Bollag, Tung, 1972, Bleakley, Tiedje, 1982, Burth, Ottow, 1983). Important results about one of possible biochemical mechanism of this phenomenon were obtained in recent studies (Shown, Tanimoto, 1991, Shown et. al., 1992). It was discovery that fungus Fusarium oxysporum and some related species have dissimillatory nitrate and nitrite reductase and a cytochrome P-450nor functioning as NO-reductase. F. oxysporum stoichiometrically converted nitrate and nitrite to nitrous oxide under conditions of reduced oxygen pressure. The purpose of present work was the investigation of capacity of various groups of soil microscopic fungi to produce nitrous oxide under different conditions in nutrient media and sterile soil. 1 MATERIALS AND METHODS The fungal cultures were isolated from different types of soils (soddy-podzolic soil, chernozems, solonchaks, brown soil and some others), roots zone of plants (tomato, cucumbers, wheat), surface of wheat seeds. The isolation was performed at 250C under aerobic conditions with Czapek and Hetchinson media. To suppress the growth of bacteria 100 mg/l streptomycin sulfate or 4 ml/l lactic acid were added to the media. The cultures were transfered to Czapek agar with chloramphenicol and streptomycin and then examined for purity from bacteria on water agar, meat-peptone agar and glucose-peptone medium and by light microscopy. The N2O producing cultures were tested on bacterial contamination also after gas chromatography measurements. The strains were maintained at slants with malt agar at 40C. The systematic determination of fungi was done on the base cultural and morphological characteristics according to recommended keys for identification. The strains were precultured in liquid Czapek medium for 5-7 days, then their mycelium was washed by 4 times by distilled water and transferred to flasks (100 ml) with liquid media (50 ml). Mycelium of fungi was grown also at Czapek agar. In this case it was transferred to the flasks without washing. The liquid media contained glucose (10,0 g), mineral salts (KH2PO4, MgSO4, KCl), microelements (Na2B4O7, CuSO4, FeSO4, MnSO4, ZnSO4, NaMoO4) and different sources of nitrogen (1,0 g) (nitrate, nitrite, peptone, ammonium and its combination) (composition was done on the base of Cove modified media, pH 7.0-7.5, Bollag, Tung, 1972). After inoculation, the strains were incubated at aerobic conditions in the media (the flasks were closed by cotton stopper), microaerobic (the flasks were closed by rubber stopper) and anaerobic conditions (the flasks were closed by rubber stopper, degassed, flushed and air replaced by argon) at 260C for 10 days. Control flasks were not inoculated and emission of nitrous oxide from control was not observed or was negligible. Each 2 days the upper space gas was analyzed by gas chromatography with detectors of thermal conductivity and electron capture. The flasks incubated at aerobic conditions were closed for 12 hours before measurements. The possible changes pH of media during cultivation of fungi and its influence on nitrous oxide release were taken in the account. In the preliminary experiments was established that noticeable emission of nitrous oxide (around 10 ng N-N2O/cm3 .hr-1 or 1.0 µg N-N2O/cm3 for 14 days) was observed at pH below 6.0 from the medium with peptone and nitrite and at pH bellow 4.0 from the medium with nitrite. Because of this the measurements of pH in the media were conducted after incubation of fungi and gas chromatography analysis. At majority of cases pH of media had not decline below these values after incubation of mycelium. The dry weight of mycelium was determined after washing 4 times at distillated water and drying at 700C for 24 hours. The repetition of flasks in the experiments were in 3 times. The strains of fungi Fusarium solani 302, F. oxysporum 11dn1, Trichoderma harzianum 18dn1, Chaetomium globosum 1dn1 (producing N2O in liquid media) were selected for inoculation in sterile soils. The seived (2 mm) samples of soils (3 g) were placed in flasks (12 ml), sterilized 3 times in an autoclave, wetted by 5.0 ml sterile distilled water or sollution of nitrate/nitrite, glucose, inoculated by fungal mycelium and closed by rubber stopper. The variants of experiment included the soddy-podzolic and chernozem soils 2 without any supplements and treatments with nitrite (2 µg N-NO-2/g), nitrate (100 µg NNO-3/g) separatly and together with 2.5 µg/g glucose. Amounts of suplements of glucose, nitrate and nitrite in the peat soil were significantly higher - glucose 10 mg/g, nitrites and nitrates (2 mg/g). The flasks were flushed by helium or argon. Fungal mycelium that was obtained on liquid Czapek medium and inoculated to sterile soddy-podzolic soil and chernozem at the rate 10 mg/g and to peat soil at the rate 2 mg /g soil. Control flasks with sterile soils were inoculated by fungal mycelium and sterilized. The flasks were incubated in 250C for 10 days and periodically the upper space gas was analyzed by gas chromatography. RESULTS AND DISCUSSION The production of nitrous oxide was tested at 62 fresh cultures of saprotrophic micromycetes belonging to 50 species of classes Zygomycetes, Ascomycetes and Deuteromycetes. Fungi do not form nitrous oxide at aerobic conditions on the all tested nitrogen sources. Not one of fungal strain released nitrous oxide on the media with ammonium, organic nitrogen (peptone or β-alanin). Many species of soil microscopic fungi of classes Deuteromycetes and Ascomycetes were capable to produce nitrous oxide at the conditions of reduced pressure of oxygen and the presence of nitrite (19 from 62 strains) and nitrite with peptone in media (23 from 62 strains). Few strains of Fusarium oxysporum and F. solani released of nitrous oxide on the medium with nitrate and nitrate and peptone. Most active producers of nitrous oxide belonged to Fusarium oxysporum, F. solani, Chaetomium globosum, Neosartorya fisheri, Trichoderma, Penicillium expansum, Talaromyces and some others species (table 1). Obtained results have increased diversity of fungal species capable to produce nitrous oxide. Activity of nitrous oxide production by fungi was in the range 0.5210 µg N-N2O/ml for 10 days (15-1244 ng N-N2O/ml/hour) and in the calculation on the biomass of mycelium - 0,2-2 µg N-N2O/ml/g/hour. Production of nitrous oxide by Fusarium oxysporum 9dn2, Chaetomium globosum 1dn1, Trichoderma 17dn1 and some other micromycetes was 2-240 µM N2O and for most active strains - 40-240 µM N2O for 7 days. Nitrite was much more suitable substrate than nitrate by using that microscopic fungi can produce nitrous oxide. The biochemical investigations are needed for the understanding why only few species of fungi from that can grow on nitrate in aerobic conditions are able to form nitrous oxide at the conditions of reduced pressure of oxygen. This phenomenon can be connected with presence of dissimilatory nitrate reductase in nitrous oxide producing fungi or opportunity of assimilatory nitrate-reductase for dissimilatory variant of the functioning. At the same time many fungal strains that have assimillatory nitrate-reductase can produce nitrous oxide on the nitrite at the conditions of reduced pressure of oxygen. Probably, these fungi have nitrite-reductase that are active in the anaerobic conditions. Comparison of activities of nitrous oxide production by microscopic fungi with denitrifying bacteria have shown that it was on several orders lower than denitrifying bacteria demonstrate (Mahne, Tiedje, 1995). Emission of nitrous oxide was not found from non-inoculated by fungi soils. The nitrous oxide release was detected from all soils inoculated by F. solani 302 and F. oxysporum 11dn1. The production of nitrous oxide by these fungi have reached 28 mkg N3 N2O/g for 10 days from soddy-podzolic soil without supplements and 89, 161 µg N-N2O/g from the soil with addition of nitrate and nitrite, correspondingly. The emission of nitrous oxide by F. solani 302 and F. oxysporum 11dn1 was not detected from chernozem’s soil without supplements of nitrate and nitrite. The N2O producing activities of Trichoderma harzianum 18dn1 and Chaetomium globosum 1dn1 were 5-10 times less than in the case of the cultures of genus Fusarium. Amounts of N2O producing by the fungi at the soil conditions were at most cases less, but similar order as from the liquid media. 4 Table 1 Nitrous oxide production by microscopic fungi in liquid media Species Nitrous oxide (µg N-N2O/ml for 10 days) Substrate ----------------------------------------------------------NO-2 | NO-2+ peptone 1* 2 | 1 2 Acremonium charticola 3dn1 Alternaria alternata 4dn1 Aspergillus alliaceus 7dn1 Aspergillus gr.flavus 6dn1 Aspergillus flavus acCo 45-8-58 A. versicolor 5dn1 Aureobasidium pllulans 8dn1 Chaetomium globosum 1dn1 C. globosum 16.02 C. globosum 1dn2 Chaetomium sp. Frt 2 Cladosporium cladosporioides 9dn1 C. oxysporum x1 Cylindrocarpon sp. 10dn1 Fusarium oxysporum 11dn1 F. oxysporum 11dn2 F. oxysporum 301 F. solani 12dn1 F. solani 302 Gliocladium fimbriatum Rp1 Humicola sp. TK-7fp25-16b Neosartirya fisheri 2dn1 Oidiodendron griseum KOM1 Penicillium expansum 13dn1 Penicillium sp. 14dn1 Penicillium sp. 15dn1 Stachybotrys atra 16dn1 Talaromyces flavus X3 Trichoderma aureoviride 17dn1 T. koningi 19dn1 T. longibrachiatum 18dn1 0.5 3.1 0.0 0.0 0.0 3.0 58.0 22.0 24.0 4.0 15.2 210.0 7.3 75.0 25.0 180.0 6.0 2.5 0.0 13.0 0.0 0.0 0.0 0.0 2.4 5.8 0.0 2.6 0.0 0.0 0.0 4.0 17.0 8.0 28.0 6.0 0.0 130.0 0.0 89.0 0.0 110.0 2.0 1.4 0.0 7.0 0.0 0.0 0.0 8.0 0.0 0.0 3.0 3.6** 0.0 4.4 0.0 2.0 0.0 3.1 24.0 40.0 0.0 5.9 3.6 99.0 11.2 6.0 4.4 1.9 2.0 1.9 3.1 3.5 2.4 9.1 6.5 6.5 1.6 2.0 3.1 16.0 2.0 2.2 2.0 5.4 0.0 3.7 3.8 124.0 8.3 4.8 1.0 1.3 2.4 4.0 2.6 2.1 2.8 6.0 6.3 * - 1- microaerobic conditions; 2 - anaerobic conditions; ** - coefficient variation of data - 10-15%. The discovery of capacity to produce N2O between many species of microscopic fungi that are widely distributed in soils and root zone of plants in liquid media and opportunity to 5 realize this activity at soil conditions allow to propose the ecological significance of this phenomenon. LITERATURE Bleakley B.H., Tiedje J.M., 1982: Nitrous oxide production by organisms other than nitrifiers or denitrifiers. Appl. Environ. Microbiol., 1982, 44, 6, p.1342-1348. Bollag J.M., Tung G., 1972: Nitrous oxide release by soil fungi. Soil Biol. Biochem. 4, p.271-276. Burth J., Ottow J.C.G. Influence of pH on the production of N2O and N2 by different denitrifying bacteria and Fusarium solani. Ecol. Bull. 1983, 35, p.207-215. Mahne I. and Tiedje J., 1995: Criteria and methodology for identifying respiratory denitrifiers // Appl. Environ. Microbiol. 61, 3:1110-1115. Shown H. and Taninmoto T., 1991: Denitrification by the fungus Fusarium oxysporum and Involvement of cytochrome P-450 in the respiratory nitrite reduction. J.Biol.Chem., 266, 17, p.11078-11082. Shoun H., Kim D.-H., Uchiyama H., Sugiyama J. Denitrification by fungi. FEMS Microbiol. Lett., 1992, 94, p.277-282. Keywords : fungi, soil, nitrous oxide, nitrite, nitrate Mots clés : champignon, sol, oxydes nitreux, nitrite, nitrate 6