Peer Review #3 of "Manure application increased denitrifying gene abundance in a drip-irrigated cotton field (v0.1)"

Abstract

Application of inorganic nitrogen (N) fertilizer and manure can increase nitrous oxide (N 2 O) emissions. We tested the hypothesis that increase in N 2 O flux from soils amended with manure reflects a change in bacterial community structure and, specifically, an increase in the number of denitrifiers. To test this hypothesis, a field experiment was conducted in a drip-irrigated cotton field in an arid region of northwestern China. Treatments included plots that were not amended (Control), and plots amended with urea (Urea), animal manure (Manure) and a 50/50 mix of urea and manure (U+M). Manure was broadcastincorporated into the soil before seeding while urea was split-applied with drip irrigation (fertigation) over the growing season. The addition treatments did not, as assessed by nextgen sequencing of PCR-amplicons generated from rRNA genes in soil, affect the alpha diversity of bacterial communities but did change the beta diversity. Compared to the Control, the addition of manure (U+M and Manure) significantly increased the abundance of genes associated with nitrate reduction (narG) and denitrfication (nirK and nosZ). Manure addition (U+M and Manure) did not affect the nitrifying enzyme activity (NEA) of soil but resulted in 39-59 times greater denitrifying enzyme activity (DEA). In contrast, urea application had no impact on the abundances of nitrifier and denitrifier genes, DEA and NEA; likely due to a limitation of C availability. DEA was highly correlated (r = 0.70-0.84, P < 0.01) with the abundance of genes narG, nirK and nosZ. An increase in the abundance of these functional genes was further correlated with soil NO 3-, dissolved organic carbon, total C, and total N concentrations, and soil C:N ratio. These results demonstrated a positive relationship between the abundances of denitrifying functional genes (narG, nirK, and nosZ) and denitrification potential, suggesting that manure application increased N 2 O emission by increasing denitrification and the population of