The Mechanism Of Exogenous Carbon Input On Nitrate Nitrogen Assimilation Rate And Denitrification Nitrogen Loss In Fluvo-aquic Soil

The North China Plain is vital for the national crop production;however,the characteristics of intensive high nitrogen（N）fertilizer input,low N use efficiency and soil N cycling with strong mineralization and nitrification,and weak immobilization and denitrification have resulted in serious soil nitrate（NO₃^-）accumulation in this area.The control of soil NO₃^-concentration is the prerequisite to prevent soil NO₃^-accumulation.Lowering NO₃^-concentration by increasing the rate of soil microbial NO₃^-immobilization is likely to play an important role in reducing NO₃^-accumulation in fluvo-aquic soil.The microbial NO₃^-immobilization rate is often very low or even negligible in fluvo-aquic soil on account of carbon（C）-limited,while,exogenous organic C input is likely to remove restriction of microorganisms to C and therefore stimulates soil microbial NO₃^-immobilization.However,the effect of exogenous organic C input on microbial NO₃^-immobilization rate and its microbial mechanism in fluvo-aquic soil are still unclear.The project used ¹⁵N tracing technique and acetylene（C₂H₂）inhibition methods to clarify the selection basis of calculation methods for microbial NO₃^-immobilization rate with exogenous organic C input and combined the above two methods with real-time quantitative PCR technology to elucidate the effect of the quantity and quality of exogenous organic C input on NO₃^-immobilization rate and the associated microbial mechanism and to reveal the influence of exogenous organic C input on denitrification N loss and nitrous oxide（N₂O）emission in a fluvo-aquic soil.Accurately quantifying the rate of microbial NO₃^-immobilization is the premise of reducing NO₃^-accumulation in cultivated fluvo-aquic soil.However,there is still a little research on the accuracy comparison of microbial NO₃^-immobilization rates that were commonly calculated by three methods including ¹⁵N pool dilution,organic ¹⁵N recovery and microbial biomass ¹⁵N recovery methods.Thus,an incubation study was conducted in a cultivated fluvo-aquic soil,and seven organic materials（two readily available C sources and five plant residues differencing in C/N ratio）addition treatments with the same C level(5 g C kg^-1)and a control treatment（without organic material addition）were set up.We investigated denitrification N loss and microbial NO₃^-immobilization rates calculated by the three methods in a cultivated fluvo-aquic soil under various organic material amendments using a combination of ¹⁵N tracing technique and C₂H₂ inhibition.The results showed that the three methods used herein resulted in similar microbial NO3^-immobilization rates for the fluvo-aquic soil amended with plant residues;In contrast,for fluvo-aquic soil amended with readily available C sources（glucose and molasses powder）,the NO₃^-immobilization rates obtained using the ¹⁵N pool dilution method were 65～100%and 47～74%higher than those estimated by the organic ¹⁵N recovery and microbial biomass ¹⁵N recovery methods,respectively.Less than 0.25%of the added ¹⁵NO₃^-was recovered as the NH₄⁺–N pool,suggesting that the rate of either the dissimilatory reduction of labeled NO₃^-to NH4⁺or re-mineralization was negligible and both the organic ¹⁵N recovery and microbial biomass ¹⁵N recovery methods produced reliable estimates of microbial NO3^-immobilization rate.The denitrification N loss rate during the whole incubation period was less than 1.8%in the fluvo-aquic soil amended with organic materials,confirming the reliability of the ¹⁵N pool dilution method.However,considering the convenience of data acquisition,the organic ¹⁵N recovery method was more accurate and saves time and effort.We also developed microbial NO₃^-immobilization-specific quality indexs that incorporate C/N ratio,contents of lignin,cellulose and hemicellulose,and p H for slowly decomposing organic materials（plant residues）,which calculated by different methods,thus,providing a direct selection basis of calculation methods for the cultivated fluvo-aquic soil microbial NO₃^-immobilization rate with exogenous organic C input.Previous studies have shown that microbial NO₃^-immobilization in cultivated soils was regulated by the quality and quantity of exogenous organic C input.However,the quantitative relationships between the quality and quantity of organic C and the microbial NO₃^-immobilization in fluvo-aquic soil are still unresolved.Thus,we set up seven organic materials（two readily available C sources and five plant residues with different C/N ratios）addition treatments each with four levels of C input(0,no C source added,control;1 g C kg^-1,low C addition level;2 g C kg^-1,medium C addition level;5g C kg^-1,high C addition level)in the fluvo-aquic soil.¹⁵NO₃^-labelling technique and real-time PCR method were used to investigate microbial NO₃^-immobilization rates under different various exogenous organic C input and bacterial and fungi abundances with and without different exogenous organic C input under a high C addition level in the fluvo-aquic soil.The results showed that there was a positive linear correlation between microbial NO₃^-immobilization rate and amount of added exogenous organic C,regardless of carbon source types in the fluvo-aquic soil.Specifically,microbial NO₃^-immobilization rates per unit of added pure C of two readily available C sources（glucose and molasses powder）,maize leaves,wheat straw and sawdust,and rice bran and rice husk were 2.05～2.16,0.77～1.13,and 0.29～0.38 mg N kg^-1d^-1,respectively.Irrespective of the amount of C input,soil microbial NO₃^-immobilization rate was significantly positively with content of holocellulose,holocellulose/total N ratio and C/N ratio.Under the addition of high C,soil microbial NO₃^-immobilization rate was significantly positively correlated with the abundances of bacterial and fungi and negatively with the ratio of bacterial/fungi.Furthermore,the content of holocellulose of added plant residues significantly affected the NO₃^-immobilization rate by affecting the bacterial abundance.In conclusion,this study elucidated the quantitative relationships between the quality and quantity of exogenous organic C and microbial NO₃^-immobilization rate and the associated microbial mechanism in the fluvo-aquic soil,thus,contributing to the development of regulation strategies to lower NO₃^-concentration and reduce NO₃^-accumulation in cultivated soils.In addition to microbial NO₃^-immobilization,denitrification in the fluvo-aquic soil is also C-limited.Thus,the input of exogenous organic C necessarily stimulates soil denitrification N loss and greenhouse gas N₂O emission.However,how to maximize the microbial NO₃^-immobilization rate while avoiding the denitrification N loss and N₂O emission in the fluvo-aquic soil remains unaddressed.Our foregoing study has demonstrated that denitrification N loss was responsible for less than 1.8%of added¹⁵NO₃^-in all treatments with high C addition level(5 g C kg^-1),indicating that denitrification N loss under exogenous C input was probably negligible in the fluvo-aquic soil.However,it was still of great importance to investigate N₂O emission pathways given that N₂O is a powerful greenhouse gas.Therefore,we investigated N₂O emission pathways,gross N-cycling rates,and associated N-cycling gene abundances in the fluvo-aquic soil under various organic material amendments using a combination of ¹⁵N tracing technique,C₂H₂ inhibition,and real-time PCR methods.We showed that increased total N₂O emissions in fluvo-aquic soil following organic material amendment was attributed to both increased nitrification-derived N₂O emissions,following increased ammonia-oxidizing bacteria（AOB）-amo A abundance,and denitrification-derived N₂O emissions,following increased nir S and decreased nos Z abundances.Increasing plant residue C/N ratio decreased total N₂O emissions by decreasing the contribution of denitrification to N₂O emissions,potentially due to higher proportions of denitrified N emitted as N₂O than nitrified N emitted as N₂O.We further proposed a novel conceptual framework for organic material input effects on denitrification-derived N₂O emissions based on the decomposable characteristics of the added organic material.For slowly decomposing organic materials（e.g.,plant residue）with insufficient available C,NO₃^-immobilization surpassed denitrification,resulting in gradual decrease in denitrification-derived N₂O emission with an increase in mineralization of plant residue C losses.In contrast,available C provided by readily available C sources（e.g.,glucose）seemed sufficient to support the co-occurrence of NO₃^-immobilization and denitrification.Overall,for the first time,we offered a microbial process perspective of N₂O emissions in fluvo-aquic soil following organic material input.The findings could facilitate the improvement of process-orientated models of N₂O emissions and the formulation of appropriate N₂O mitigation strategies for crop residue-amended fluvo-aquic soil.