Effects Of Nitrogen Addition On Soil Carbon And Nitrogen Mineralization And Microbial Regulation Mechanism In A Pinus Tabulaeformis Forest

Atmospheric nitrogen?N?deposition has accelerated in the last several decades due to anthropogenic activities,and is significantly influencing soil carbon?C?and N cycling processes of forest ecosystem on a global scale.Understanding the characteristics of soil C and N mineralization and the microbial regulation mechanism of forest ecosystems under the background of N deposition is of great significance for maintaining soil sustainable development and predicting global change.However,the research in this area in the Loess Plateau artificial forest ecosystem is still insufficient.In this study,soil samples were collected from an artificial Pinus tabulaeformis forest located in Loess Plateau?China?to which N at four different concentrations was added(0[N0],3[N3],6[N6],and 9[N9]g N m^–2 y^–1)for 5 years.By field sampling,artificial climate chamber incubating and metagenomics techniques?Geo Chip 5.0?,we systematically studied the effects of N addition on C and N mineralization characteristics,enzyme activities and kinetic parameters,microbial community functioning,and the temperature sensitivities of C and N mineralization and enzyme kinetic parameters.Our study cleared the response of soil C and N content,enzyme activity and C and N mineralization in a soil profile?0-100 cm?to different N addition levels,clarified the effect mechanism of N addition on different functional enzymes catalytic performance and microbial functional gene community,and synthesized plant-soil-microorganism factors to reveal the microbial regulation mechanism of soil C and N mineralization process under different N addition levels,which provided scientific basis for deeper understanding of the soil C and N transformation process in this area.The main results of our study were as follows:?1?N addition improved C and N nutrient contents in different soil layer,but there was a threshold effect of N addition.Higher than 6 g N m^-2 a^-1,it may have an adverse effect on soil C nutrients.Overall,N addition significantly increased the contents of soil organic C?SOC?,dissolved organic C and N?DOC,DON?,nitrate?NO₃^–-N?,microbial biomass C and N?MBC,MBN?,and soil C/N in 0-50 cm soil layers,but had no significant effects on soil p H,total N,total P,and available P contents,litter and fine root biomass.NO₃^–-N,DON and MBN concentrations were consistently elevated with increasing N addition levels,and reached the peak value in N9 treatment.SOC and DOC concentrations,MBC and the soil C/N ratio initially increased and then decreased with increasing N addition levels,and reached the highest values in N3 or N6 treatment,respectively.N addition has a weak effect on the C and N nutrient contents in the 50-100 cm soil layer.In all treatments,except for p H value,other soil factors decreased with the increasing soil depth.?2?N addition increased C and N transformation rates in different soil layers,but had different effects on the temperature sensitivity.In the 0-50 cm soil layer,low N addition?N3and N6?significantly increased soil C,N mineralization rate and N₂O emission rate by9.73%,16.84%,and 8.73%,respectively,when the amount of N addition was higher than6 g N m^-2 a^-1,it would have a weak inhibitory effect on C mineralization and N₂O emission rate.This change was related to soil N limitation-saturation status.N addition had no significant effect on C and N transformation rates in the 50-100 cm soil layer.In addition,the study of C and N transformation temperature sensitivity in topsoil?0-10 cm?found that the Q₁₀ values of N₂O emission ranged between 1.16-1.27;the Q₁₀ values of C and N mineralization ranged between 1.66-1.78 and 1.63-1.80,respectively,which are lower than the global average levels?2.30 and 2.21?.N addition significantly reduced the Q₁₀ value of C mineralization and had no significant effect on N mineralization Q₁₀ value,but significantly increased the Q₁₀ value of N₂O emission,indicating that the increase in N deposition may offset the C loss caused by warming to some extent and accelerate soil N loss in the context of climate warming.?3?N addition had different effects on different functional enzyme activities,kinetic parameters,and the temperature sensitivity,showing that N addition significantly stimulated oxidase,C-and P-acquisition enzyme activities of the 0-20 cm soil layer,but significantly suppressed N-acquisition enzyme activities.The V_max values of?-glucosidase?BG?and?-D-cellulosidase?CBH?increased with increasing N addition levels,and the K_m values decreased and increased with N addition,respectively.Overall,the catalytic efficiency of C-acquisition enzyme was improved.The response of N-acquisition enzyme V_max and K_mvalue to N addition was affected by temperature.At low temperature??15°C?,N addition reduced the V_max and K_m values of N-acetyl-?-glucosaminidase?NAG?and leucine amino-peptidase?LAP?;while at high temperature?35°C?N addition significantly increased the K_m values of both.Overall,N addition significantly inhibited the catalytic efficiency of NAG and LAP.N addition significantly increased the V_max and K_m values of soil P-acquisition enzyme,but had no significant effect on the catalytic efficiency.The results showed that N addition regulated the apparent activities of different functional enzymes by differentially affecting the V_max and K_m values of soil enzymes.N addition increased the Q₁₀values of V_max and K_m of all soil enzymes,and decreased the Q₁₀ value of V_max/K_m.However,significant differences were only observed in BG,NAG and LAP,indicating that the temperature sensitivity of N-acquisition enzyme was more sensitive to N addition.?4?Based on functional gene microarray Geo Chip 5.0,our study revealed the effect and mechanism of N addition on the microbial functional gene community,founding that N addition significantly changed community composition of microbial functional genes.Low N addition?N3 and N6?had no significantly effect on the total relative abundance and diversity of genes community but significantly increased the relative abundance of specific genes for the labile C degradation?such as starch,chitin,pectin?,NH₃ cycle?such as ure C,nir A,and nrf A?,nitrification?amo A?and denitrification?nor B?.These positive effects were related to the increase in DOC,NO₃^–-N,and MBC.While above the N addition threshold of6 g N m^-2 a^-1 would decrease the relative abundance and diversity of microbial functional genes community,and this negative impact was correlated with reduced DOC and MBC.The results indicated that,unlike the N-rich ecological system,the regulation strategies of N addition to microbial functional genes in our study area was mainly mediated by changes in soil C and N nutrients,and has no significant correlation with p H.?5?Microbial functional gene abundance and composition play an important role in soil C and N mineralization.In the process of SOC mineralization,path model analysis found that N addition changed microbial C-degradation genes and SOC-decomposing enzyme activities indirectly by affecting soil properties,and finally regulated CO₂ emission.Among these factors,microbial C-degradation genes have the greatest influence on CO₂emission.Specifically,the increase in CO₂ emission rates may be mainly related to the higher abundance of functional genes for labile C degradation under low N treatment?N3 and N9?,whereas the lower CO₂ emission rates may be mainly related to lowering abundance of functional genes for labile as well as recalcitrant C degradation under high N treatment.In the process of soil N transformation,microbial N functional genes were more important than soil parameters for explaining changes in N transformation within our study site.Further analysis by stepwise multiple regression showed that ammonia-oxidizing archaea?amo A-AOB?and MBC were the key factors related to net N nitrification;ure C,nir K,and MBC were the key factors related to net N mineralization;and nar G and nir S were the key factors related to N₂O emission.Results mentioned above indicated that different N deposition levels may have different effects on soil nutrient content,enzyme activities and microbial functional characteristics,and thus differently changed soil C and N transformations processes.The current amount of N deposition(2.2 g N m^-2 a^-1)in the study area may improve soil C and N nutrient status,increase soil enzyme activity and microbial functional gene abundance,and thus accelerate soil C and N mineralization rates and improve soil nutrient status.Considering the importance of microbial community and functional parameters for soil nutrient conversion,they should be incorporated into the current global C and N cycle and climate models to improve the prediction accuracy of these models.