Nitrogen Transformation Associated Microbial Mechanisms In Typical Grassland Soils In Inner Mongollia

Grassland,as one of the largest terrestrial ecosystems in the world,accounts for up to 40.5%of the world’s terrestrial land area,and 41.7%of China’s total land area.Grassland ecosystems are important for food production to support the growing world population.It is reported that about 70-90%of the N ingested by the grazing animal is returned to the grassland soils in animal excreta,particularly in urine.Therefore,it is important to study the N cycling in the grassland ecosystem for its contributions to climate change and for developing sustainable grassland ecosystems.Grassland soils are great sinks of CH₄.Methane is oxidized by the methane monooxygenase（AMO）enzyme,which is encoded by the pmoA genes of methane oxidizing bacteria（MOB）.Ammonia oxidation is the first and rate-limiting step of nitrification.It has been shown that methane oxidizers and ammonia oxidizers are evolutionarily linked.Therefore,strong interactions exist between ammonia oxidization and methane oxidization,while these effects of grazing on the interactions are poorly understand.Effects of grazing on soil microbes are by means of eating,trampling and excreta.That is why we choose the three management practices:enclosing,grazing and mowing in this study.This research firstly focused on the effects of pasture management practices on nitrification and denitrification related microbes.Then the effects of long-term grazing as well as gradient grazing on soil general bacteria,active nitrifiers and nitrification activity were studied.This was followed by a study on the microbial pathways for N₂O emissions in sheep excreta-treated soils.Lastly,the effects of grazing on interactions between ammonia oxidization and methane oxidation were investigated.（1）Soil samples were collected from a semiarid grassland ecosystem in the Xilingol region,Inner Mongolia,where long-term management practices including free-grazing,different periods of enclosure from grazing and different frequencies of mowing were established.The AOA amoA copies ranged from 5.99x108 to 8.60×108 while those of AOB were in the range of 3.02x107 to 4.61×107.The abundance of AOA was substantially higher in the light grazing treatment than in the mowing treatments.The quantity and intensity of DGGE bands of AO A varied with pasture management.In stark contrast,the AOB population abundance and community structure remained largely unchanged in all the soils irrespective of the management practices.Higher gene abundance and greater intensity of DGGE bands of nirS and nosZ genes under the enclosure treatments suggested greater stimulated denitrification.The ratio of nosZ/（nirS+nirK）is higher in the mowing treatments than in the free-grazing and enclosure treatments,possibly leading to more complete denitrification.Correlation analysis indicated that inorganic nitrogen content and soil moisture were the two main soil environmental variables that influenced the community structure of nitrifiers and denitrifiers.（2）The distribution of soil general bacterial and nitrifying communities was significantly affected by gradient grazing.Light to mediate grazing significantly increased the abumdance of bacteria and archaea.Some functional microbes were specifically enriched after long term grazing,such as Nitrospirae（phylum）to Nitrospira（class）in the lightly grazed samples and Chromatiales（order）to Nitrosococcus（genus）in the heavily grazed soils.DNA-SIP and Miseq sequencing analysis suggested that AOA and AOB respectively dominated nitrification in lightly and heavily grazed soils.（3）N₂O emissions from urine were significantly higher than from dung,ranging from 0.12 to 0.78 kg N₂O-N ha’1 in the urine patches.The N₂O emissions were significantly related to the bacterial amoA and nirK gene abundances.It was autotrophic nitrification that dominated N₂O production in the low urine-N rate soils,whereas it was denitrification（including nitrifier denitrification and heterotrophic denitrification）that dominated N₂O production in the high urine-N rate soils.Nitrifier denitrification was responsible for most of the N₂O emissions in the dung-treated soils.This study suggests that nitrifier denitrification is indeed an important pathway for N₂O emissions in grazed grassland ecosystems.（4）Strong interactions between ammonia oxidization and methane oxidation exist in grassland soils.The effects of urea addition on methane oxidization presents to be low-promoting and high-repressing,while the effects of methane addition on nitrification activity depends on the amount of urea addition.Inhibited effects of methane addition on nitrification activity were found under low levels of urea addition,while no significant effects of methane addition on nitrification activity were found under high levels of urea addition.Compared with the zero grazing treatment,grazing significantly decreased the methane oxidation potential.The community structure of active MOB is simple,comprising mainly of type la Methylobacter.In grazed soils,over 99%of active MOB are affiliated with Methylobacter.