The Mechanism Of ANAMMOX Bacteria's Morphological Structure Changes Regulating Physiological Metabolism Under Salinity Stress

Anammox bacteria are a type of chemical autotrophic bacteria that can use nitrite as an electron acceptor to convert ammonia into nitrogen under hypoxic conditions,therefore playing an important role in the nitrogen cycle.The anammox process has become the most promising wastewater biological denitrification process due to its advantages of high efficiency,low energy consumption,and low operating cost.The processes related to anammox are also increasingly used to treat the high-nitrogen wastewater such as filtrate,aquaculture wastewater and chemical pharmaceutical wastewater.However,the high concentration of salt in the actual wastewater inhibits the activity of anammox bacteria,thereby hindering the practical application of the process.Although there have been studies analyzing the inhibition of salinity on anammox bacteria and its effect on the performance of anammox reactor.However,these studies did not get consistent conclusion.Therefore,the inhibitory mechanism of salinity on anammox bacteria remains to be further studied,especially the adaptive strategy of anammox bacteria under salinity stress requires scientific exploration.Cell morphology is an important influencing factor of cell function and metabolic behavior.At present,there are few studies on the mechanism of anammox bacteria cell morphology and structure regulating their physiological metabolism to adapt to salinity stress from the perspective of cell morphology and function regulation.This study took anammox bacteria as the object,and studied morphological changes and physiological metabolic responses of anammox bacteria from initial inhibition to late adaptation under the stress of salinity(Na Cl).The adaptation mechanism of anammox bacteria to salinity was revealed from the perspective of cell morphology regulation.The main research contents and results of this paper are as follows:(1)In order to explore the influence of salinity on the morphology and structure of anammox bacteria,Synchrotron Radiation was used to reconstruct the anammox bacteria under salinity stress.It was found that the volume of anammoxosome became smaller and the folds of the membrane inwardly sag deepened.Transmission electron microscopy imaging results of the ultrastructure of anammox bacteria in different stress periods are consistent with the results of three-dimensional reconstruction.A statistical analysis of the relative size and cell fractal dimension of anammox in the results of transmission electron microscopy using Image J showed that the relative volume of anammox was reduced from the initial 59.5% to 36.5% of the fractal dimension of cell morphology under salinity stress,and the the fractal dimension of cell morphology gradually increased from the initial 1.8064 to 1.8200 as the stress time increased.The cells shrank and the degree of morphological irregularity gradually increased.The statistical analysis of the cell size of anammox bacteria by flow cytometry found that the cell morphology also gradually decreased.(2)Under salinity stress,the dnitrogen removal performance of the reactor decreased first and then gradually recovers after 2-3 generation cycles,which indicated that inhibition of moderation salinity on anammox bacteria can be quickly restored,and anammox bacteria have higher salt tolerance potential.The enzyme activity of Hydrazine dehydrogenase(HDH),NADH: Ubiquinone oxytonase(Nuo),Cytochrome C reductase(bc1)and ATP content in different stress periods were detected.Both the enzyme activity and ATP increased sharply at the beginning of salting,then gradually decreased,and finally recovered to close to the initial value under long-term stress.(3)In order to explore the potential role of ladder lipids in salinity stress,the ladder lipids in the anammox membrane were extracted.The vesicles were assembled,and the related membrane properties of assembled vesicles were tested.The relative proportion of ladder lipids with a carbon chain length of 20 increased from the initial92.1% to 94% under salinity stress,indicating that the thickness of the cell membrane was increasing.The Young's modulus of the vesicles decreased from the initial87.1±9kpa to 73.5±4kpa on the 3rd day,and then gradually recovered.These results indicated that the mechanical properties of the membrane decreased and the density decreased in the early stage of salting,which increase membrane permeability,promote proton pumping and related enzymes activity.But in the later stage of salting,the mechanical properties gradually recovered,the density of Anammoxosome membrane increased,which can resist high osmosis pressure and prevent cells from losing water.The p H transmembrane equilibrium speed of vesicle accelerated on the 3rd day and then gradually recovered,which indicated that the vesicle transmembrane proton transfer speed increased in the early stage of salting,promoting the synthesis of ATP.These results further suggested that anammox bacteria could resist the initial salinity stress by enhancing energy metabolism(4)Salinity inhibited the abundance and diversity of microbial populations.The species with salt tolerance potential can be screened out through inhibition,and the microbial community structure can be replaced in the high salt environment.Under salinity stress,Planctomycota,Proteobacteria,Chlorocurve,and Streptococcus were the dominant phyla in the reactor.Unclassified Candidatus Brocadiaceae and Candidatus Kuenenia belong to An AOB were detected.Among them,unclassified Candidatus Brocadiaceae was the dominant genus of An AOB,and its abundance increased from37.31% initially to 62.05% onthe 18 th day under salinity stress,indicating unclassified?Candidatus?Brocadiaceae has high salt tolerance potential(5)Coupled analysis of the morphological structure of anammox bacteria with the results of material energy metabolism and ladder lipid membrane performance,it was found thatthat the morphological structure was significantly correlated with material energy metabolism and anammox membrane properties.Therefore,under salinity stress,anammox bacteria may control the changes of cell morphology and structure through central metabolism,thereby regulating the material and energy metabolism activity in harsh environments,improving cell survival rate,and resisting the inhibition of salinity on cell physiological activity.At the same time,the regulation of the central metabolic network and changes in the activity and abundance of related membrane proteins have led to adaptive coordination of lipid metabolism pathways,and have improved membrane components to couple changes in cell morphology and energy metabolism.