| Large amounts of saline wastewater are produced with the process of urbanization,industry and agriculture.More than 5%of the global wastewater was accompanied by high salinity.Saline wastewater usually contains high content of organic carbon,nitrogen and phosphorus.The salinity in saline wastewater poses a great threat to the biological wastewater treatment systems,resulting in nutrient accumulation in the effluents.And the excessive nutrient not only brings difficulties to the standard discharge of industrial saline wastewater,but also leads to eutrophication in water bodies.Therefore,effective strategies for saline wastewater treatment is necessary to break through the bottleneck of sustainable industry and to alleviate the threat to ecosystems.Biological nitrogen removal is a topic in studies of wastewater treatment to attenuate the stresses of environmental pollution and water scarcity.Processes of nitrification-denitrification based on ammonia oxidation are the mainstream strategy for biological nitrogen removal in current wastewater treatment plants.But it becomes vulnerable in severe environments,particularly,nitrogen removal performance is greatly inhibited under saline conditions with notorious nitrite and nitrate accumulated.Other alternative processes have disadvantages including sensitivity to environmental conditions,unclear metabolic pathways or difficulty regulating.So finding effective nitrogen removal approaches in saline wastewater treatment is still a hot topic to attenuate the stresses of environmental pollution and water scarcity.In the process of ammonium assimilation,ammonium is incorporated into 2-oxoglutarate to produce glutamine catalyzed by glutamate dehydrogenase(GDH)or by glutamine synthetase/glutamate synthase(GS/GOGAT).It has been estimated that the interconversion of ammonium and organic nitrogen quantitatively dominates the biogeochemical nitrogen cycling,especially in marine environments.Furthermore,this ammonium uptake by heterotrophic bacteria is implied as a valuable strategy for nitrogen recovery.Although ammonium assimilation has been well studied at the cellular and genetic levels,the significance of this metabolic pathway in saline wastewater treatment has not been appreciated.Therefore,the construction of the ammonium-assimilating microbiome can shed a light on developing a desirable approach to synchronous removal and recovery of nitrogen for refractory saline wastewater treatment.In this study,one marine bacterium with the ability to form sedimentary granular biofilms was selected and engineered to construct an efficient ammonium-assimilating microbiome.Firstly,the nitrogen metabolic pathway of the functional microbe was analyzed and verified by genome sequencing and the 15N isotope labeling test.The effects of different factors such as initial concentrations,carbon to nitrogen ratios and different salinity on the nitrogen and phosphorus removal performance of the strain were demonstrated.Secondly,a halophilic ammonium-assimilating microbiome was constructed from the functional bacterium through self-assembly with environmental microbes.The COD,ammonium,total nitrogen and total phosphorus removal performance during the long-term operation verify the feasibility of the ammonium-assimilating microbiome in saline wastewater treatment.The functional gene abundance,nitrogen balance and the 15N isotope labeling test were used to verify the nitrogen conversions in the ammonium-assimilating microbiome.And the dominant ammonium assimilation in this microbiome provided an alternative strategy to improve the nitrogen metabolism in saline wastewater.Finally,the optimization of ammonium assimilation by carbon to nitrogen ratios and the effect of salinity on the functional stability of this microbiome were investigated to reveal the key factor to regulate the treatment performance of the ammonium-assimilating microbiome.The main results of this study were as follows.Furthermore,understanding the microbial interactions and their response to different C/N ratios and different salinity is crucial to proving the adaptability and applicability of the ammonium-assimilating biosystem.(1)One marine bacterium with the ability to form sedimentary granular biofilms was selected.According to the nitrogen functional genes in the genome,this strain could utilize ammonium through ammonium assimilation.Genes coding for urease,nitrate reductase and assimilatory nitrite reductase were also detected.In phenotypic analysis,ammonium was removed without the detection of nitrite or nitrate production.The balanced TN between the supernatant and the biomass demonstrated that no gaseous nitrogen was produced in the system.Furthermore,the 15N abundance in cells showed an increase,while neither 15N2 or 15N2O was detected in the 15N isotope labeling test.Ammonium utilization was impervious to the addition of the ammonia oxidation inhibitor.This strain had higher removal efficiency of ammonia nitrogen under relatively low initial ammonium concentration(25 mg/L and 50 mg/L)and low salinity(1-3%).The process of nitrogen assimilation was accompanied by the utilization of carbon sources.When the carbon to nitrogen ratio is 25,the strain showed the best nitrogen and phosphorus removal performance.(2)An ammonium-assimilating microbiome was constructed through self-assembly of this strain with environmental microorganisms,followed by the bottom-up design of the engineering microbiome.This ammonium-assimilating microbiome achieved efficient nutrient removal performance that the average removal efficiencies of ammonium,total nitrogen and total phosphorus were 88.4%、82.6%、98.7%、84.5%,respectively.Meanwhile,the accumulation of neither nitrite nor nitrate was detected in the ammonium-assimilating microbiome.The dominance of ammonium assimilation in nitrogen utilization of the microbiome was also verified by the TN balance,as well as the 15N isotope labeling test.The abundances of glnA in the ammonium-assimilating microbiome were relatively high at an average of 3.91×105 copies per ng DNA,1.80-fold higher than that in the nitrifying microbiome.Remarkably,the gene abundances of both amoA and nxrA(representing ammonia oxidation and nitrite oxidation)remained extremely low,proving that nitrification is negligible in the ammonium-assimilating microbiome.Furthermore,the analysis of the microbial community demonstrated that heterotrophs were enriched in the ammonium-assimilating microbiome,which maintained the functional stability of ammonium assimilation in the microbiome.(3)The optimization of carbon to nitrogen(C/N)ratios(10-30)on the treatment performance of the ammonium-assimilating microbiome was investigated.Results revealed that the increase in C/N ratios significantly improved the nitrogen and phosphorus removal efficiencies.With sufficient biomass,higher than 86%ammonium and higher than 73%phosphorus were removed when the C/N ratios were higher than 25.Ammonium assimilation dominated the nitrogen metabolism even under the relatively low C/N ratio,evidenced by the extremely low abundances of nitrification functional genes.Notably,different C/N ratios did not significantly change the bacterial community structure of ammonium-assimilating biosystems,although heterotrophic microbes with different preferences were enriched under different carbon loads.(4)In hypersaline wastewater under the salinity from 3%to 7%,the treatment performance and bacterial community of the ammonium-assimilating microbiome were investigated.High salinity significantly hindered nitrogen and phosphorus removal efficiency in ammonium-assimilating biosystems,but the high salinity did not change the direction of nitrogen conversions.The ammonium removal efficiency decreased from 60.8%at the salinity of 3%to 29.1%at the salinity of 7%,while the phosphorus removal efficiency dropped from 60.7%to 33.4%at the salinity of 3%and 7%,respectively.The average concentration of nitrite and nitrate was lower than 1 mg/L and 2 mg/L,respectively.Furthermore,the balanced TN and gene abundances verified the dominance of assimilation in nitrogen metabolic pathways and the exclusion of nitrification in all biosystems.High salinity also stimulated the production of extracellular polymeric substance(EPS)and changed the microbial community.The average concentration of EPS increased from 45.36 mg/g MLSS to 85.91 mg/g MLSS at the salinity of 3%and 7%,respectively.With the increase in salinity,several halophilic genera were enriched in the bacterial community.Meanwhile,the abundances of genes related to membrane transport and carbon metabolism increased with salinity.The relative abundances of ammonium-assimilating functional genes glnA and gdhA remained stable,which proved that the ammonium assimilation pathway remained stable in the microbiome.Overall,the ammonium-assimilating microbiome maintained functional stability of nitrogen metabolism and robust sludge properties in hypersaline wastewater.(5)The decrease of salinity(from 3%to 0.5%)had a significant impact on the nitrogen removal efficiency and nitrogen metabolism pathway in the ammonium-assimilating microbiome.When the salinity decreased from 3%to 1.5%,the ammonium assimilation dominated the metabolic pathway of nitrogen metabolism.The removal efficiency of ammonium decreased slightly with the decrease of salinity,while the removal efficiency of total nitrogen was consistent with that of ammonium.When the salinity continually decreased to 1%and 0.5%,the accumulation of nitrite and nitrate occurred in the microbiome,indicating the presence of nitrification.In addition,the removal efficiency of total nitrogen was significantly lower than that of ammonium.When the salinity recovered to 3%,the process of ammonium assimilation gradually dominated the nitrogen metabolism.Furthermore,with the decrease in salinity,the particle size of sludge increased,and the sedimentation of sludge and production of EPS decreased.The decrease in salinity also significantly changed the microbial community structure.Nitrosomonas,the typical nitrifying microorganism AOB was detected in the system when the salinity decreased to 0.5%.Results of nitrogen functional genes demonstrated that the abundance of ammonium-assimilation genes was significantly higher than that of nitrification functional genes under the relatively high salinity from 1.5%to 3%.Under low salinity(from 0.5%to 1%),the abundance of nitrification function genes increased significantly.It could be concluded that salinity was the key factor to maintain the stability of ammonium assimilation in the microbiome.To sum up,this study creatively proposed the method of constructing an environmental microbiome from the functional microorganism.An ammonium assimilating microbiome with efficient nitrogen removal and recovery ability was successfully constructed,which proved the feasibility of applying ammonium assimilation into the environmental microbiome,and provided an alternative strategy for saline wastewater treatment.This novel process could be applied in other fields of artificial nitrogen management,including terrestrial saline wastewater treatment,control of coastal water pollution,and biological remediation of saline soils. |
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