Environmental Distribution,enrichment Culture And The Role Of Intermediate Product Effects Of Complete Ammonia Oxidizers

The discovery of complete ammonia oxidizers(comammox)broke the past knowledge of the traditional nitrification process by directly oxidizing ammonia nitrogen to nitrate with lower energy requirements.Since the discovery of comammox,a lot of research has been conducted on the environmental distribution,enrichment culture and physiological and biochemical properties of comammox,but due to the short time of discovery,there are still many unknown aspects about the potential ecological niche differentiation and physiological and biochemical properties of comammox in the natural environment.The Yellow River estuary is a special area of sea-land interaction with complex and variable environmental processes.Studying the distribution of nitrifying bacteria throughout the region is essential to understand the influence of environmental factors on comammox,and also provides a reference for setting environmental parameters for laboratory enrichment.Hydroxylamine(NH2OH),as an intermediate product of comammox,is a key part of the study of its physiological and biochemical properties,so it is important to study the mechanism of hydroxylamine effect on comammox to further investigate its physiological and biochemical properties.In this thesis,the whole process nitrifying bacteria were studied in two aspects:natural ecosystem distribution and laboratory research.Firstly,the research was conducted in three habitats(Wang Yuan Lou inlet,wetland and Yan Lake)of the nearshore ecosystem of the Yellow River inlet to investigate their distribution patterns and bacterial population structure in the natural ecosystem.Then two sequential batch membrane bioreactors(SBBR),R1 and R2,were operated to carry out laboratory enrichment culture based on the research.After successful enrichment,R1 was kept under the same enrichment conditions and R2 was added with hydroxylamine to investigate the mechanism of intermediate products on comammox by analyzing the nitrogen conversion,comammox activity,microbial community structure and abundance changes during the experiment.The main research results were obtained as follows:(1)Comammox was prevalent in aquatic ecosystems and sediments.The presence of full-scale nitrifying bacteria was detected at all stations in the nearshore ecosystem of the Yellow River inlet,but the gene abundance of comammox varied greatly among the three habitats,with the highest abundance of comammox in the bottom sediment of Yan Lake and the lowest abundance of comammox in the sediment of the Wang Yuan Lou inlet.(2)Environmental factors significantly influenced the distribution of comammox.comammox abundance was negatively correlated with pH and salinity,and positively correlated with TC,TN,NH4+and NO3-.comammox abundance was less correlated with NH4+than AO A and AOB,indicating a higher affinity of comammox for ammonia nitrogen.(3)Based on the preliminary research,the laboratory enrichment was carried out under oligotrophic environment with pH 7 ± 0.1 and low ammonia nitrogen concentration,and the ammonia nitrogen removal rate in the reactor was stabilized above 80%after 120 days,and the gene copy number of comammox in R1 and R2 reached 2.28 × 107 ± 2.61 × 106 and 2.75 × 107 ± 1.24.× 106 copies/g dry weight,which was more than 20 times of the inoculated sludge,indicating that comammox had been effectively enriched at this time.(4)During the hydroxylamine addition stage,the nitrogen conversion and microbial community structure of the two reactors showed significant differences.ammonia nitrogen removal rate and effluent nitrate concentration in R2 reactor were lower than those in Rl,and the activity of comammox showed a decreasing trend from 28.3±1.42 mgN/(Kg·d)in the enrichment stage to 14.6 ± 1.02 mgN/(Kg.d);the relative abundance of comammox in R1.The gene abundance of comammox in the reactor was 2.51 times higher than that of R2;the relative abundance of Nitrospira was 3.99 times higher than that of R2.It indicates that the long-term addition of hydroxylamine inhibits the activity and growth of comammox.(5)During the hydroxylamine addition stage,the changes of extracellular polymer secretion by microorganisms in the reactor were analyzed,and it was found that the amount of protein secreted by comammox in R1 and R2 reactors were 79.76 ± 4.91 and 155.19±0 mg/g VSS,respectively.thus,it is clear that hydroxylamine addition would have an effect on the secretion of extracellular polymer by comammox,stimulating The addition of hydroxylamine affected the secretion of comammox extracellular polymers,stimulating the secretion of more proteins by comammox,which led to enhanced sedimentation performance of activated sludge,but also led to a decrease in mass transfer efficiency,which in turn led to a decrease in denitrification performance due to insufficient substrate uptake.(6)After the addition of hydroxylamine produced a continuous and stable inhibitory effect on the whole nitrifying bacteria,the addition of hydroxylamine was stopped,and the sex and abundance of comammox gradually recovered.After stopping the addition of hydroxylamine,the effluent water quality of R2 reactor gradually recovered,and at this stage,the activity of comammox was 91.1±2.73 mgN/(Kg·d),which was 6.24 times higher than that at the stage of hydroxylamine addition;the abundance of comammox gene was 3.36×109±2.44 × 108 copies/g dry weight,which was 34.67 times higher than that at the stage of hydroxylamine addition.The relative abundance of Nitrospira was 42.84%,which was also significantly higher than that of hydroxylamine.This indicates that the inhibitory effect of hydroxylamine on comammox is reversible and can be gradually restored after stopping the addition.