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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.
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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
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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.
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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
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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
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