Biases Of Acetylene And Octyne As Selective Inhibitors For Determining Soil Nitrification And Denitrification Rates

Nitrification and denitrification play an important role in soil nitrogen cycling.They are not only important pathways for nitrogen loss in soil,but also the major sources of nitrous oxide emissions.Alkynes?octyne and acetylene?,as selective inhibitors,are widely used to study soil nitrification and denitrification processes.Octyne inhibition technique is the most commonly method to distinguishthe relative ammoxidation potential between ammonia-oxidizing bacteria and ammonia-oxidizing archaea.The acetylene inhibition technique is the most commonly used method for determining soil denitrification rate.At present,the biases of octyne and acetylene as selective inhibitors in determining soil nitrification and denitrification rates have not been well quantified,and the mechanisms involved are not clear yet.In order to study the bias of octyne inhibition technique in distinguishing the ammonia oxidation rate between bacteria and archaea,the pure strains of typical ammonia-oxidizing bacteria and ammonia-oxidizing archaea were used to track the dynamics of nitrogen-containing gases?N₂O and NO?during the ammoxidation process under different octyne concentrations.The bias of octyne in determining soil ammoxidation rate was quantified.The results show that:octyne has the ability to catalyze the oxidation of NO into NO₂,as similar as acetylene.In addition,octyne is insoluble in water and can not completely inhibit the ammoxidation of bacteria,which leads to bias in using octyne to distinguish the relative contributions of bacteria and archaea to the production of N₂O during ammoxidation.The octyne inhibition technique seriously underestimates 0.25-1 times of the soil ammoxidation rate.As the concentration of octyne??20uM?increases,the biases caused by octyne inhibition technique increases.The bias caused by octyne-catalysed oxidation of NO into NO₂ accounts for45%of the total bias value,while the bias caused by incomplete octyne inhibition of the bacteria ammoxidation activity accounts for 55%of the bias value.Our results indicate that when the octyne concentration is 10uM,the octyne inhibition technique has the minimal bias in distinguishing the relative amount of N₂O produced during the ammoxidation of bacteria and archaea.In order to quantitatively evaluate the bias of acetylene inhibition technique in determining soil denitrification rate,this paper quantifies the bias of acetylene in determining the denitrification rate using the helium/nitrogen substitution-direct quantitative nitrogen method.The results show that:the acetylene inhibition technique seriously underestimates 5-26 times of the denitrification rate,which does not reflect the increasing trend of denitrification rate with the increasing soil water content.The bias value of the acetylene inhibition technique increases with the increase in soil nitrate and water contents.There are two main sources of biases of the acetylene inhibition technique.One source is the acetylene-catalysed oxidation of NO,which on average accounts for 60%?31%-79%?of the total bias of the acetylene inhibition technique.Another source of bias is the incomplete acetylene inhibition of N₂O reduction,which on average accounts for 40%?14%-71%?of the total bias of the acetylene inhibition technique.Our results indicate that,under the aerobic conditions,the acetylene inhibition technique has a large bias in determining soil denitrification rate,so it is not recommended to use acetylene inhibition technique to determine the soil denitrification under aerobic conditions,especially for soils with high nitrate and water contents.In summary,the biases of both acetylene and octyne inhibition techniques should be considered when using them to study the nitrification and denitrification processes in soils.The results of this paper help us to clarify the biases range and applicable conditions of the acetylene and octyne inhibition techniques,which is helpful for applying them to determine the rates of denitrification and ammonium oxidation under aerobic conditions and interpreting the data more reasonably.