Effects Of Earthworm On Asymbiotic Biological Nitrogen Fixation Under Nitrogen Deposition

Biological nitrogen fixation,nitrogen mineralization and nitrogen deposition are the three major sources of available nitrogen in forest ecosystems.Although the nitrogen fixation efficiency of asymbiotic biological nitrogen fixation is often lower than that of symbiotic nitrogen fixation,it occurs in a broader range of ecosystems.Asymbiotic nitrogen-fixing microorganisms could be found in floor litter and soil in most forest ecosystems.Asymbiotic biological nitrogen fixation,approximately in the range of 1 to 20 kg N ha-1 yr-1,is the main source of exogenous nitrogen input in ecosystems lacking nitrogen fixation plants.There may be some tradeoff between nitrogen mineralization and asymbiotic nitrogen fixation in ecosystems.The increasing nitrogen deposition will further change the balance between nitrogen mineralization and asymbiotic biological nitrogen fixation.The activities of earthworms could regulate the process of nitrogen mineralization,and it has also been found that there are diverse nitrogen-fixing microorganisms and greater nitrogenase activity in the intestines of earthworms.To explore the effects of earthworms on asymbiotic nitrogen fixation in the context of nitrogen deposition is on the cutting edge in studing the response and feedback of soil biota to global changes.Given that earthworms have a very low carbon to nitrogen ratio?around 4.0?,nitrogen is particularly important for their survival and growth.The extent to which earthworms may depend on organic nitrogen mineralization and asymbiotic biological nitrogen fixation,and the extent to which nitrogen deposition may alter these processes,are still unknown.The forest ecosystems in the temperate-subtropical transition zone possess the characteristics of both the north and south biomes.To explore the relationships between nitrogen deposition,earthworms and soil asymbiotic biological nitrogen fixation process in the transition zone ecosystems is of great significance for understanding the change pattern of soil biota ecological functions in the context of global changes.Furthermore,the traditional understory-simulated nitrogen deposition ignored the process of canopy nitrogen retention and transformation,and thus failed to manifest the virtural effect of nitrogen deposition on forest ecosystems.Based on these considerations,this study raises the following scientific questions: Does the activity of earthworms affect the asymbiotic biological nitrogen fixation process? Does nitrogen deposition reduce the effect of earthworms on asymbiotic biological nitrogen fixation,and how do the earthworm effects may differ in systems with canopy-and understory-simulated nitrogen deposition? In this study,forest soils and litters with different treatments of nitrogen deposition were sampled in 2018 from the field manipulation experimental platform of "Canopy-Understory Simulated Nitrogen Deposition" which was built in Jigongshan Mountain,Xinyang,Henan province in 2013,including treatments of field control?N0?,canopy addition of 25 kg N ha-1 yr-1?CN₂5?and understory addition of nitrogen 25 kg N ha-1 yr-1?UN₂5?.Then,a microcosm experiment,including control?CK?,earthworm addition in the non-sterilized system?W?,system with sterilized soil and litter?S?,and earthworm addition in the sterilized system?W+S?,was set up using the three types of soil and litter,respectively.The main research contents are as follows: determining soil nitrogenase activity by acetylene reduction method;analyzing the composition and content of microbial phospholipid fatty acid?PLFAs?to indicate the characteristics of soil microbial community;quantifying the asymbiotic nitrogen fixation by ¹⁵N₂ labeling;and examing the mineralization process of soil organic matter by measuring the changes of soil carbon and nitrogen content.Thereby,the influences of earthworms on the balance of "nitrogen mineralization and asymbiotic nitrogen fixation" in different nitrogen deposition systems were explored.The main findings are as follows:?1?The soil nitrogen content in Jigongshan broadleaved deciduous forest ecosystem was relatively low,and there was observable asymbiotic nitrogen fixation in the forest floor?soil and litter?.The acetylene reduction method showed that the ethylene generation rate of the control forest soil and litters?N0 system?was higher than that in the CN₂5 and UN₂5 systems,indicating that the nitrogen deposition has a certain inhibition effect on the floor asymbiotic nitrogen fixation in the Jigongshan forest.?2?The ¹⁵N₂ labeling experiment showed that the abundances of 15 N in earthworm bodys were not significantly increased,indicating that earthworms themselves did not utilize nitrogen derived from asymbiotic biological nitrogen fixation.We considered that earthworms may get enough nitrogen from organic matter decomposition in the Jigongshan broadleaved deciduous forest ecosystem.?3?The ¹⁵N₂ labeling experiments showed that the mean 15 N abundance of N₂ O released by CN₂5 system was 8.9 ‰,while that released by the sterilized CN₂5 system was as high as 254.6 ‰,far greater than the mean natural 15 N abundance of N₂O?-0.25‰?,indicating that CN₂5 system had obvious Asymbio--tic nitrogen fixation.The presence of earthworms significantly reduced the 15 N abundance of N₂ O in CN₂5 system,indicating that earthworms inhibited the asymbiotic nitrogen fixation process.On the contrary,the 15 N abundance of N₂ O released by UN₂5 system was not significantly increased regardless of whether or not the systme was sterilized,indicating that the asymbiotic biological nitrogen fixation was very weak in UN₂5 system.?4?The inorganic nitrogen in the three types of forest soils with different treatments of nitrogen deposition was mainly ammonium nitrogen?NH4+-N?,and there were no significant differences of NH4+-N concentration between these soils.Soil sterilization significantly increased the NH4+-N concentration in the control forest soil?N0 soil?and CN₂5 soil,and the presence of earthworms further increased it.These results indicated that the N0 and CN₂5 systems may possess more mineralizable organic matter than the UN₂5 system.While,earthworm activities can significantly promote the mineralization process of organic matter,and thus inhibit asymbiotic biological nitrogen fixation.?5?The data of phospholipid fatty acid?PLFAs?showed that the soil microbial community was significantly different in the three systems with different nitrogen deposition.On the one hand,compared with N0 and CN₂5 systems,both the soil microbial biomass and bacterial stress index of UN₂5 system were higher.This indicated that the soil microbial community of the UN₂5 system was in a relatively "stationary phase" with the high microbial biomass but limited bacterial activity.This may reflect a depletion of readily mineralizable carbon in the UN₂5 system.The long-term higher input of inorganic nitrogen in the UN₂5 system may increase soil microbial biomass but at the same time consume a large amount of available organic carbon,and thus exacerbating the carbon limitation of soil microorganisms.Such carbon limitation may be an important reason behind the observed weak asymbiotic nitrogen fixation in the UN₂5 system.On the other hand,earthworm activity significantly reduced the bacterial stress index of the UN₂5 system and increased the bacterial biomass,but did not exert significant effects in the N0 and CN₂5 systems.These results futher suggested that the available resources in the UN₂5 system are relatively poor,while earthworm activities can alleviate the resource constraints of soil bacteria.In conclusion,the floors in both N0 and CN₂5 systems had significant asymbiotic biological nitrogen fixation,but earthworm activity may significantly inhibit it probably by promoting organic matter mineralization.However,the understory-simulated nitrogen deposition?UN₂5?may deplete mineralizable carbon and thus inhibit bacteria activity and weaken the asymbiotic biological nitrogen fixation;in addition,earthworm activity would further inhibit the asymbiotic biological nitrogen fixation process.Therefore,we considered that the understory-simulated nitrogen deposition cannot illustrate the virtual effect of nitrogen deposition on soil biota and the related carbon and nitrogen processes in Jigongshan forest ecosystem.Further studies are needed to focus on the changes of asymbiotic nitrogen-fixing microorganisms in response to the nitrogen deposition and earthworm activities,so that the underlying mechanisms would be fully revealed.