Emission of greenhouse gases and microbial biodiversity in soils of agricultural interest. Effect of nitrogen fertilisation

Résumé

SUMMARY In agricultural soils, the application of inorganic nitrogen (N) fertilisers leads to the interaction of multiple factors and processes which are mainly associated with changes in soil physicochemical properties, emission of greenhouse gases and microbial ecology. Although N is an essential nutrient for plant growth, increased application of N-fertilisers in agriculture has altered the natural N cycle, which result in many environmental, ecological and human health impacts. Among them, N-fertilisation may lead to an increase in the emission of greenhouse gases, acidic deposition and eutrophication. After application of an N-fertiliser, the microbial processes of nitrification and denitrification are the main responsible of the reactions driving the conversion of ammonia (NH4+) and nitrate (NO3-) to the release of the greenhouse gas nitrous oxide (N2O) into the atmosphere, respectively. Combating the negative impacts of increasing N2O fluxes poses considerable challenges and will be ineffective without incorporating microbial regulated N2O processes into mitigation strategies. Although previous studies had shown individual relationships between N-fertilisation and soil biotic and abiotic parameters, an integrated study relating the form of the N fertiliser with differences in N2O emission, changes in soil physicochemical properties, alterations in the abundance of the genes involved in N2O production and reduction, and effects on bacterial diversity had not been reported. To address these questions, the N-fertilisers urea ([CO(NH2)2] ammonia (NH4)2SO4 and nitrate (KNO3) were chosen to amend an agricultural Cambisol soil from Vega de Motril (Granada, Spain). Tomato (Solanum lycopersicum) and common bean (Phaseolus vulgaris) were used as representative vegetable plants. Soils, cultivated or not, were kept under greenhouse conditions and fertilised at an N rate of 260 kg N ha-1. Unfertilised soil was used as a control. Uncultivated soils were incubated for 3 years and cultivated soils for 4 consecutive harvests of about 4 months each. During that time, the soils were watered once a week to reach 80% water filled pore space (WFPS). The concentration of the fertilisers was determined regularly and when required the soil was supplemented with the corresponding N-fertiliser to reach the initial N-fertilisation rate. Soil abiotic variables pH, NH4+, NO3-, total carbon (TC), total N (TN) and total organic carbon (TOC) were determined by spot sampling during incubation. The production of N2O was regularly determined by gas chromatography. Soil samples were taken to determine the total abundance of a) soil bacteria (16SB) and archaea (16SA) by quantitative PCR (qPCR) of the 16S rRNA gene, respectively; b) ammonia oxidising (AO) bacteria (AOB) and archaea (AOA) by qPCR of the corresponding amoA gene, respectively, and c) denitrification genes by qPCR of the napA, narG, nirK, nirS, norB, nosZI and nosZII genes. Variations in the relative abundance of the bacterial OTUs were determined after pyrosequencing of the 16S rRNA gene. To analyse the distinct effect of N-fertilisation and soil depth, urea, ammonium and nitrate were applied to the soil. N2O production and the abundance of N-cycling genes were determined along the 20-cm layer of the arable topsoil. Variations in N2O emissions along the soil profile were dependent on the type of N-fertiliser and the soil depth-related dissolved oxygen content. Also, N-gas emissions correlated with the abundance of the nitrifying and denitrifying communities in the soil. While N2O production by nitrification was dominant in the 0- to 10-cm soil horizon, denitrification was the main driver of N-gas production in the 10- to 20-cm depth. The nosZ gene was the most sensitive to soil depth-related dissolved oxygen content. To determine the effect of N-fertilisation on the a) soil physicochemical characteristics, b) N2O emission, c) changes in the abundance of the bacterial and archaeal communities, d) abundance of the nitrifying and denitrifying guilds and e) variations in bacterial diversity, urea, ammonium and nitrate were applied to the soil cultivated or not with tomato and common bean. The study included the bulk and rhizosphere soil of the plants. Fluxes of N2O emission showed a peak about 2 weeks after N-fertilisation both in cultivated and uncultivated soils. The cumulative N2O emission was higher in cultivated than uncultivated soils, and higher in the soil cultivated with common bean. Regardless of the presence or the absence of the plants, on a yearly basis, urea produced the higher cumulative emission followed by ammonium and, finally, nitrate. Differences in N2O emissions were associated to a distinct ratio of the genes involved in the production and reduction of N2O. Simultaneous application of high water moisture content and inorganic N-fertiliser was required for maximum N2O production. Decreases in N2O production during 3-year incubation for uncultivated soils and 4 consecutive harvests for cultivated soils were associated to increases in the nosZ gene abundance. N-fertilisation decreased the abundance of the total bacterial and archaeal communities in uncultivated soils and increased their abundance in the cultivated soils. These results were associated to the soil carbon content. The amoA AOA was more abundant than the amoA AOB gene in the rhizosphere soil and, on the contrary, the abundance of the amoA AOA was lower than that of the amoA AOB in the bulk soil. The denitrification genes were more abundant in the bulk soil. N-fertilisation decreased the number and the relative abundance of bacterial OTUs in soils cultivated or not with tomato and common bean plants; this effect was more severe in the rhizosphere soil. N availability mainly determined the changes in the structure of the bacterial community in bulk and even more in the rhizosphere soil, and the bacterial community became less diverse or dominated by a small group of OTUs. After N-fertilisation, dominant and rare OTUs decreased in the rhizosphere while only the rare OTUs vary in the bulk soil. To explore the relative contribution of nitrification and denitrification to N2O production after 3-year fertilisation with ammonium or nitrate, the 15N tracer technique was used. In the ammonium-treated soil, N2O originated from nitrification almost equally that from denitrification and emission from the nitrate-treated soil derived mostly from denitrification. The higher abundance of the nosZI gene in the soil treated with nitrate was consistent with the highest 15N2 enrichment. To build a model to understand the relative importance of each analysed biotic and abiotic variables and bacterial biodiversity as drivers of N2O emission, combined random forest (RF) and structural equation modelling (SEM) analyses were run using the dataset derived from this study. The results show that N2O emissions were mainly controlled by the biotic (amoA AOA. amoA AOB, napA, nirK, norB, nosZI genes and a set of 16 bacterial OTUs) than the abiotic (NH4+ and NO3- contents) variables. To quantify the importance of bacterial and fungal denitrification post nitrate application, single and combined application of bacterial (streptomycin) and fungal (cycloheximide) growth inhibitors were used. Although bacteria and fungi almost equally contributed to soil N2O production, bacteria dominated over that of fungi during the first 2-3 days post nitrate application. After that time, the situation reversed and the production of N2O by fungi came to dominate that of bacteria. Investigation of the effects of the single and combined application of the urease inhibitor N-(n-butyl) thiophosphoric triamide (NBPT) and the nitrification inhibitor 3,4 dimethylpyrazole phosphate (DMPP) on ammonia (NH3) volatilisation and the abundance of the nitrifier and denitrifier communities have also pursued in this study. The application of the urease inhibitor NBPT reduced NH3 volatilisation and did not affect the bacterial and archaeal abundance, nor that of the nitrifiers, but reduced the abundance of denitrifiers at 80% WFPS. DMPP, alone and in combination with NBPT, increases NH3 volatilisation and the abundance of bacteria, archaea and nitrifiers in the soil. Regardless of the moisture conditions, DMPP and to a lower extent DMPP + NBPT, increases the gene copy number of the norB- and nosZ-bearing denitrifying communities, which indicates that DMPP, somehow, induces the expression of the, at least, the norB and nosZ denitrification genes.