Taxonomic and denitrifying bacteria communities associated with the interception of nitrate leaching by carbon amendment in the subsoil

Nitrate leaching is severe in greenhouse agriculture where excessive nitrogen is often applied to maintain high crop productivities. Carbon amendment in the subsoil, where denitrification is limited by the availability of carbon, might mitigate nitrate leaching. In this study, we investigated the effects of carbon amendment in the subsoil on nitrate leaching and the emission of greenhouse gases (CH 4 and N 2 O) emissions using a soil column experiment. Diversity and abundance of total and nirS-, nirK- , and nosZ -type denitrifying bacteria were investigated by high throughput sequencing of PCR amplicons and quantitative real-time PCR. The amounts of nitrate leaching were >39% less in the treatments with carbon amendment than in the non-amended control without fertilization or in treatments fertilized by two doses (1600 or 3200 kg N ha - 1 ) of ammonia or nitrate. No effects of carbon amendment on the emissions of CH 4 or N 2 O were observed. The total N content in the subsoil zone with carbon amendment increased from 20.74% to 70.54%. Strikingly, the abundance of nirS , nosZ and 16S rRNA was higher in the treatment than the corresponding controls while no significant effects were detected for nirK . Carbon amendment rather than fertilization was the primary factor that influenced the community composition of the three denitrifying bacterial communities and explained 14%, 10%, and 4% of the variation in the community of nosZ, nirS , and nirK, respectively. Decreased alpha-diversity and increased variability in beta-diversity were observed for the carbon amended treatment for total and denitrifying bacteria. On average, genera such as Anaerovorax, Pseudobacteroides, Magnetospirillum, Prolixibacter, Sporobacter, Ignavibacterium, Syntrophobacter, Oxobacter, Hydrogenispora, Desulfosporomusa, Mangrovibacterium, and Sporomusa were enriched more than seven times in soil amended with carbon. In summary, carbon amendment in the subsoil mitigated nitrate leaching and increased the nitrogen pool by possible activation of denitrifying and anaerobic bacterial populations.

of carbon amendment in the subsoil on nitrate leaching and the emission of greenhouse gases (CH 4 and N 2 O) emissions using a soil column experiment. Diversity and abundance of total and nirS-, nirK-, and nosZ -type denitrifying bacteria were investigated by high throughput sequencing of PCR amplicons and quantitative real-time PCR. The amounts of nitrate leaching were >39% less in the treatments with carbon amendment than in the non-amended control without fertilization or in treatments fertilized by two doses (1600 or 3200 kg N ha -1 ) of ammonia or nitrate. No effects of carbon amendment on the emissions of CH 4 or N 2 O were observed. The total N content in the subsoil zone with carbon amendment increased from 20.74% to 70.54%. Strikingly, the abundance of nirS , nosZ and 16S rRNA was higher in the treatment than the corresponding controls while no significant effects were detected for nirK . Carbon amendment rather than fertilization was the primary factor that influenced the community composition of the three denitrifying bacterial communities and explained 14%, 10%, and 4% of the variation in the community of nosZ, nirS , and nirK, respectively. Decreased alpha-diversity and increased variability in beta-diversity were observed for the carbon amended treatment for total and denitrifying bacteria. On average, genera such as Anaerovorax, Pseudobacteroides, Magnetospirillum, Prolixibacter, Sporobacter, Ignavibacterium, Syntrophobacter, Oxobacter, Hydrogenispora, Desulfosporomusa, Mangrovibacterium, and Sporomusa were enriched more than seven times in soil amended with carbon. In summary, carbon amendment in the subsoil mitigated nitrate leaching and increased the nitrogen pool by possible activation of denitrifying and anaerobic bacterial populations.

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However, the manuscript can be downloaded and accessed as a PDF.  The effects of carbon amendment on nitrate leaching accumulation (a), soil total nitrogen in the subsoil zone (b), soil CH4 concentration(c), and soil N2O concentration (d) (n=3).
Significant difference (p<0.05, t-test) between carbon amendment and non-amended control was indicated by different letters. Carbon: carbon amendment; CK: non-amendment.
Non-fertilizer: no fertilizer; NH4+Low: with ammonia fertilization at 1600 kg N ha-1; NH4+High: with ammonia fertilization at 3200 kg N ha-1; NO3-Low: with nitrate fertilization at 1600 kg N ha-1; NO3-High: with nitrate fertilization at 3200 kg N ha-1.  indicate the phylotype with significantly higher relative abundance in the treatment with the 8 carbon amendment soil. Relative abundance in permile was indicated by color. ND: not detected. The percentage behind OTU indicated the similarity between the representative sequence of the OTU to the reference sequences. Accession number was in the bracket. Carbon: carbon amendment; CK: non-amendment. Non-fertilizer: no fertilizer; NH4+Low: with ammonia fertilization at 1600 kg N ha-1; NH4+High: with ammonia fertilization at 3200 kg N ha-1; NO3-Low: with nitrate fertilization at 1600 kg N ha-1; NO3-High: with nitrate fertilization at 3200 kg N ha-1.