Enrichment And Characteristics Of Soil Ammonia-oxidizing Archaea And Its Mechanism Of High Ammonia Tolerance

Ammonia-oxidizing archaea?AOA?play an important role in the Earth's nitrogen cycle.However,their contributions to the ammonia oxidation in farmland and other roles playing in the ecosystems remain unclear.Obtaining thaumarchaeotal strains are essential to reveal the physiological roles of different AOA phylotypes.AOA enrichment or isolation is extremely difficult and time consuming?average 2 to 3 years?.After more than ten years of efforts,only about30 AOA cultures have been obtained.The insufficiency of AOA strain limits our understanding their roles playing on ecology,physiology,and underlying biochemistry in different environments,which urge us to obtain more AOA strains from environments.In this study,we designed a two-step strategy for the rapid enrichment of Nitrosocosmicus–like AOA from soils.During the first step,a combination of kanamycin and ampicillin was chosen as the selective stress for bacteria and quartz sands were used as the attachment of AOA cells;only after 40-75 days cultivation,AOA enrichments with abundance>20%were obtained.During the second step,a combination of ciprofloxacin and azithromycin that found to be able to penetrate biofilms and kill the bacterial cells inside the aggregate was chosen as the selective stress for the following cultivation;only after one-time cultivation,AOA enrichments with high abundance?>90%?was obtained.Basing on this strategy,three AOA strains were obtained from agricultural soils only after 90-150 days cultivation.Phylogenetic analysis suggested these AOA belong to the soil group I.1b Thaumarchaeota and are closely linked to the genus Nitrosocosmicus.Here,we provided a new strategy for rapid enrichment of high abundance of Nitrosocosmicus-like AOA from soil,which gives a new solution to the AOA enrichment and cultivation in a short period.The mechanism of"two-step strategy"was found as:?1?the combination of ampicillin and kanamycin has the ability to promote the formation of biofilm by AOA,which is beneficial to its attachment and growth on quartz sand,?2?the combination of ciprofloxacin and azithromycin can effectively inhibit the secretion of extracellular polysaccharides,which suppresses the biofilm formation and then bacterial growth.In this study,an AOA strain that obtained from vegetable soil was named as Candidatus Nitrosocosmicus agrestis.Cells of Ca.N.agrestis are irregular sphere and usually embedded in a thick extracellular matrix.Ca.N.agrestis was able to grow under pH ranging from 6.0 to8.0,and the maximum specific growth rate(?_max)is 0.033 h^-1,optimum temperature 37°C,optimum salinity 0-0.2%.Ammonia-oxidizing activities were still observed at 200 mM NH₄⁺?>1500?M of free ammonia?and 50 mM NO₂,but very sensitive to nitrapyrin?NP?.Urea could be used as the sole energy source but exogenous organics had no significant effect on the ammonia oxidation.Genomic analysis suggested that,Ca.N.agrestis encodes genes for the ammonia oxidation,carbon fixation,urea hydrolysis,amino acid synthesis and some other metabolic pathways.Besides,the genome also encodes a full set of genes that responsible for polysaccharide metabolism and secretion,suggesting the probable production of extracellular polymeric substances?EPS?.In addition,the genome of Ca.N.agrestis contains a copy of low substrate affinity ammonium channel Amt-2 and several genes encoding subcellular carbonic anhydrase CA-?and CA-?-2.Differently,only?-1 type of CA that localized to the periplasm was reported to be found in other AOA groups.Ca.N.agrestis and other AOA of the genus Ca.Nitrosocosmicus is usually resistant to high ammonia.But found that for a single cell of Ca.N.agrestis,high ammonia did not significantly inhibit its ammonia oxidation,but significantly inhibit the protein synthesis efficiency.This study revealed tha the AOA of the genus Ca.Nitrosocosmicus all have DME channels which suspected as polyamine exporter.Therefore,we speculated that the mechanism of ammonia tolerance may be the synthesis of polyamines and then the excretion of polyamines through the DME transporters,and the energy consumed in this process reduces the amount of protein synthesized.The mechanism of ammonia tolerance was confirmed at transcriptional level by the relative expression of related genes under different ammonia nitrogen conditions.