(718a) N2O Production by Fungal Denitrification in a Semiarid Soil

1. Introduction

 The increasing nitrous oxide (N₂O) concentration in the atmosphere causes concern due to its contribution to global warming.¹ Soils, especially agricultural soils, contribute approximately half of the world¡¯s anthropogenic N₂O emissions and currently this source of N₂O represents 2.4% of the European release of anthropogenic derived greenhouse gasses (GHG).^2,3 Furthermore, N₂O is also directly or indirectly involved in destruction of stratospheric ozone.⁴ Modern agriculture must strive to mitigate N₂O emissions by cultivated soils.

The researches have shown that N₂O is released during the microbial nitrification and denitrification process, in principle.⁵ Soil heterogeneity permits the coexistence of aerobic and anaerobic zones which allow organisms in the same soil aggregate to function simultaneously, and nitrification and denitrification can take place at the same time.^6,7  Bcteria and fungi are the two most important microbes for emitting N₂O from agrarian soil. And both of them have the genetic potential to use organic and inorganic sources of N.^8,9 Although the basic mechanism of N₂O formation in soils is well known, there is not a better knowledge of the contribution of these microbes above.

Bacteria has been regarded as the unique microbe to denitrification for a long time, ^10,11 but fungi were found to exhibit denitrifying activities in recently.^12-14 Actually, bacteria denitrify rapidly and completely, however it need a strict condition of dissolved oxygen (DO). In contrast, fungi can simultaneously perform denitrification under microanaerobic or aerobic conditions while bacteria.

In this laboratory study, we want to know the influence of different microbes to produce N₂O. Meanwhile we also identify the N₂O production pathways by use of cycloheximide and streptomycin, inhibitorsof fungal and bacterial

Figure 1 The mechanism of denitrification and nitrification

2. Experimental

2.1. Soil and Reagents

Soil: surface (0-20 cm) clay loam soil was collected, arable but without plants, in April 2007 from ¡°El Enc"ªn¡± field station, near Alcal"¢ de Henares (Madrid, Spain) (latitude 40º 32¡¯N, longitude 3º 17¡¯W), in the middle of the Henares river basin. The soil was gently broken down by hand after transport to the laboratory, meanwhile it air-dried at room temperature during three days. Then it was sifted through a 2.5 mm sieve, and stored in plastic bag until use. Compost: Urban waste (Madrid, Spain). Inhibitor: streptomycin (Sigma-Aldrich, purity: 95%); cycloheximide (Sigma-Aldrich, purity: 94%).

Gravimetric moisture contents for the columns were derived from the relationship between wet weight of the soil column and the dry weight of the soil column. Water filled pore space (WFPS) was calculated by dividing the volumetric water content by total soil porosity. Total soil porosity was calculated according to the relationship: soil porosity = (1- soil bulk density/2.65), assuming a particle density of 2.65 g cm^-3. Bulk densities were calculated from the volume of soil in the cores.

2.3. The application of biocides to soil

Weighed 2g of soil in vial (20ml, sample:1-16). Prepared stock solution: the concentration of streptomycin was 3 mg/ml, solubility of cycloheximide was 1.5 mg/ml so the solution prepared should have dissolved it. Application of the different treatments will be carried out within the vial and these ones will be capped and left for few hours. The headspace will be sample by means of a syringe and this sample transferred to a smaller vial before being analyzed by gas chromatography.

The protocol established for the following experiments is described. This was based on the results obtained from the preliminary experiments. Deal with the effect of the application of biocides to soil (bactericide/fungicide) on N₂O production.

Table 1 The application of biocides to soil without glucose. (sample: 1-8)

C: Cycloheximide, S: Streptomycin

Table 2 The application of biocides to soil with glucose. (sample: 9-16)

G: Glucose

3. Results and Discussion

3.1. The effect of the application of biocides to soil without glucose

Table 3 The resulting concentrations of sample 1-8

Figure 2 The effect of biocides on the N₂O emission

The figure 2 shows the effect of both biocides on the fluxes, with and without acetylene. Because of little N and C in the soil and in both cases it is not clear there is inhibition when adding the biocides as the fluxes increased as compared with the blank (no added biocide). The addition of acetylene produced an increase on the fluxes in all the treatments. This could be evidence of the production of N₂ as acetylene blocks the last of denitrification. This could suggest that some of the biocides were being used by the microorganisms as a source of carbon and nitrogen or that materials leaking from the dead cells were used by the microorganisms.¹⁵ The results must be looked at carefully as there is evidence of acetylene being used as a carbon source.¹⁶

3.2. The effect of the application of biocides to soil with glucose

Table 4 The resulting concentrations of sample 9-16

Figure 3 The effect of biocides on the N2O emission with glucose

Figure 3 shows inhibition of the fluxes when using cycloheximide in the glucose treatment. The bottle with streptomycin produced an increase in the fluxes whereas the mixture of both biocides did not produce a significant effect. It showed that the streptomycin didn¡¯t work in this condition and it was used as nitrogen source by some fungal.

Figure 4 The effect of biocides on the N2O emission with acetylene and glucose

Figure 4 shows mush larger fluxes when adding acetylene compared to no acetylene addition, and an increase in the fluxes was observed with the addition of streptomycin. Because streptomycin have abundant nitrogen and it can be easily translated to N₂O by microorganisms. And this result also confirms that fungal and bacteria out-of-run mixing with acetylene and He.

4. Conclusion

Results of this study suggest that the occurrence of fungal denitrification is of ecological significance as N₂O is the dominant gaseous product in this semiarid soil. As fungi have the ability to perform denitrification and O₂ respiration simultaneously in a range of O₂-stress conditions, the potential exists for fungi to produce N₂O in a wider range of soil aeration conditions than bacteria. Fungi are widely distributed in soils and water, hence the potential exists for fungi to make a significant contribution to the global N₂O budget.

Keywords: Denitrification; Fungi; Bacteria; Streptomycin; Cycloheximide; Nitrous oxide

References

[1] Hansen J, Greenhouse gas growth rates [J], Proc. Natl. Acad. Sci., 2004, 101(46):16109-16114.

[2] Church J A and Gregory J M, The Scientifie Basis [R], Intergovernmental Panel on Climate Change (IPCC), Cambridge University, UK. 2001.

[3] Avrahami S, Effect of soil ammonium concentration on N₂O release and on the community structure of ammonia oxidizers and denitrifiers [J], Appl. Environ.Microbiol, 2002, 68 (11):5685-5692.

[4] Dyominov, Contribution of natural and anthropogenic factors to the long-term changes of the Earth¡¯s ozone layer at the end of the XX century [J], Izv. Atmos. Ocean. Phys., 2005, 41:43-55.

[5] Renault P and P Stengel, Modelling oxygen diffusion in aggregated soils: I. Anaerobiosis inside the aggregates [J], Soil Sci. Soc. Am., 1994, 58:1017-1023.

[6] Stevens R and R J Laughlin, Cattle slurry affects nitrous oxide and dinitogen emissions from fertilizer nitrate [J], Soil Sci. Soc. Am., 2001, 65:1307-1314.

[7] Kuenen J G and Robertson L A, Combined nitrification denitrification process [J], FEMS Microbiol Rev., 1994, 15(2):109-117.

[8] Merrick M J and Edwards R A, Nitrogen control in bacteria [J], Microbiol. Rev., 1995, 59: 604-622.

[9] Marzluf Ga, Genetic regulation of nitrogen metabolism in the fungi [J]. Microbiol Mol Biol Rev, 1997, 61:17-32.

[10] Takaya N. and Shoun H, Nitric oxide reduction, the last. step of the fungal denitrification by Fusarium oxysporum is obligatorily mediated by cytochrome P450nor [J], Mol. Gen. Genet., 2000, 263:342-348.

[11] Anderson J and K H Domsch, Quantification of bacterialand fungal contributions to soil respiration [J], Arch. Mikrobiol., 1973, 93:113-127.

[12] Johnson C, Measuring bacterial and fungal substrate-induced respiration in dry soils [J], Soil Biol. Biochem., 1996, 28:427-432.

[13] Laughlin R J, Determining nitrogen-15 in ammonium by producing nitrous oxide [J], Soil Sci. Soc. Am., 1997, 61:462-465.

[14] Ruzicka S, D Edgerton, M Norman, and T Hill, The utility of ergosterol as a bioindicator of fungi in temperate soils [J], Soil Biol. Biochem, 2000, 32:989-1005.

[15] Badalucco L, Activity and degradation of streptomycin and cycloheximide in soil [J], Biol Fertil Soils, 1994, 18:334-340.

[16] Dendooven L, Carbon and nitrogen dynamics in a grassland soil with varying pH: effect of pH on the denitrification potential and dynamics of the reduction enzymes [J], Soil Biology and Biochemistry, 1998, 30:359-367.