N₂O Uptake In The Processes Of Nitrogen Fixation And Denitrification Of Root Nodule Endophytes （Frankia） For Tree Species Of Asuarina Sp.

Nitrous oxide（N₂O）is one of the most important greenhouse gases,leading to global warming and depleting the ozone layer.The study on the uptake of N₂O by endophytic bacteria from non-leguminous nitrogen-fixing tree species is helpful for in-depth understanding of nitrogen-fixing tree species in forest systems.The role of N₂O absorption can also provide a scientific basis for the estimation of net N₂O emissions and the formulation of emission reduction strategies.This thesis takes casuarina root nodules in the subtropical area as the research object.The root nodule is isolated and purely cultured by sectioning to obtain Frankia strain.The morphology of liquid culture and solid culture is observed,and the internal structure of the endophyte is identified by optical microscope and electron microscope.Explore the biological characteristics of the endophyte through different media types,carbon and nitrogen sources,p H and salinity tolerance.The nitrogenase activity was determined by the acetylene reduction method.The process of Frankia denitrification and its products were explored and quantified by adding NO₃^-.The ¹⁵N₂O isotope labeling method and the acetylene inhibition method were used to verify the way of reducing N₂O with the assimilatory and dissimilatory on Frankia.The above research aims to provide a more in-depth understanding of Frankia and reveal its N₂O uptake in the process of nitrogen fixation and denitrification,and provide a scientific basis for understanding the role of nitrogen-fixing tree species in N₂O uptake in the forest system and accurately assessing N₂O emissions.The main findings are as follows:（1）Frankia hyphae have transversal branches,cysts and vesicles.The liquid culture of Frankia is flocculent and flakes sinking to the bottom or suspended in the culture medium,showing the white flesh of litter.It grows best in QMOD medium（protein content 16.58±0.632μg/m L）,followed by BAP₁（protein content 13.03±0.147μg/m L）.When glucose was used as the single carbon source,the endophyte had the best growth（protein content 17.31±5.478μg/m L）,followed by Tween 80（protein content 12.23±4.241μg/m L）.In the cultivation of different nitrogen sources,casein hydrolysate was used as the single nitrogen source to grow best（protein content 20.23±1.228μg/m L）,yeast extract（protein content 9.06±0.287μg/m L）and ammonium chloride（protein content）8.12±0.596μg/m L)followed by.At p H 6.5-7.0,Frankia is the most suitable for growth（protein content7.66±0.601-7.71±0.756μg/m L）,too much alkali and acid are not conducive to the growth of the Frankia.The growth rate of 2%Na Cl is the fastest（protein content 10.43±0.318μg/m L）,and the amount of growth decreases when it is higher or lower than 2%Na Cl.Frankia grows best under microaerobic conditions,and the dry weight of mycelium is 0.567±0.205 mg.Frankia has nitrogenase activity,and the maximum nitrogenase activity is 7.42±0.607μmol/mg C₂H₄ at 0h.The maximum nitrous oxide reductase activity for 18h was 0.194±0.014 mmol/L ¹⁵N₂.（2）Frankia has a denitrification effect in an anaerobic environment.Under anaerobic culture conditions,the liquid and headspace gas after centrifugation of the culture at different times were measured by adding NO₃^-,and a series of products were measured such as NO₃^-,NO₂^-and N₂O.The concentration of NO₃^-decreased with the increase of time,from the initial 135±2.085 mg/L to99±0.180 mg/L,and then stabilized.With the increase of time,the concentration of NO₂^-first increased and then decreased.Nitrite nitrogen reached the maximum accumulation amount of only0.177±0.030 mg/L in 41 h,and then tended to 0.The N₂O concentration first increased and then decreased with time,reaching the maximum accumulation amount of 0.249 mg/L at 54 h.Simultaneously quantitatively determine the functional gene abundance of 6 denitrification-related enzymes in different periods,including nitrate reductase（nar G）,nitrite reductase（nir K,nir S）,nitric oxide reductase（nor B）,and nitrous oxide Reductase（nos Z）and nitrogenase（nif H）.Whether at 26h or 54h,the expression of the six key denitrification genes was not synchronized.The transcription level of nitrite reductase gene is significantly higher than that of nitrate reductase gene,nitric oxide reductase gene and nitrous oxide reductase gene.The expression of the former is more than twice as high as that of the latter,which will result in a small amount of rapid conversion and accumulation of nitrite,while nitric oxide reductase and nitrous oxide reductase cannot reduce all NO and N₂O to N₂.The difference in the expression of nitrogenase in different periods indicates that Frankia may continue to reduce N₂O and N₂ by nitrogenase.（3）Frankia has two major enzyme systems to reduce the N₂O to N₂ with the assimilatory and dissimilatory.In the ¹⁵N₂O addition experiment,when no inhibitor（acetylene）is added,Frankia will produce ¹⁵N₂ from 0 h under aerobic and anaerobic conditions.The concentration of ¹⁵N₂ for aerobic treatment andδ¹⁵N is higher than the ¹⁵N₂ concentration andδ¹⁵N of the anaerobic treatment.δ¹⁵N is significantly greater than 0 under the two oxygen conditions.In addition,both the ¹⁵N₂ concentration andδ¹⁵N increased first with the increase of time under aerobic and anaerobic conditions,andδ¹⁵N both reached a peak value of 226.051±3.8722‰and 224.564±1.6311‰at 18 h,and then showed a downward trend.In addition,the abundance of ¹⁵N₂ under aerobic conditions at the four moments was greater than that under anaerobic conditions,indicating the presence of nitrous oxide reductase to reduce N₂O to N₂.Therefore,there is a coupling process of assimilatory and dissimilatory to reduce N₂O to N₂ under the experimental conditions.With the addition of inhibitor（acetylene）,Frankia produces ¹⁵N₂ from 0h under aerobic and anaerobic conditions.The ¹⁵N₂ concentration andδ¹⁵N of the aerobic+inhibitor（acetylene）treatment are higher than the ¹⁵N₂ concentration andδ¹⁵N of the anaerobic+inhibitor（acetylene）treatment,andδ¹⁵N is significantly greater than 0 under the two treatment conditions.And under anaerobic+inhibitor（acetylene）and aerobic+inhibitor（acetylene）conditions,the ¹⁵N₂ concentration andδ¹⁵N both increase first with the increase of time,andδ¹⁵N both reach a peak at 18h（225.835±2.1462‰and 224.379±1.0277‰）,and then showed a downward trend.However,the ¹⁵N₂ concentration of aerobic+inhibitor（acetylene）treatment reached a peak at 18 h,and then showed a downward trend,while the ¹⁵N₂ concentration of anaerobic+inhibitor（acetylene）treatment reached a peak at 6 h,and then decreased trend.The difference is that under anaerobic+inhibitor（acetylene）and aerobic+inhibitor（acetylene）conditions,δ¹⁵N both reach a peak at 18 h.Therefore,,there is an assimilatory process to reduce N₂O to N₂ under the experimental conditions.Therefore,in summary,Frankia has two major enzyme systems to reduce the N₂O to N₂ with the assimilatory and dissimilatory under the experimental conditions.