Enrichment And Optimization Of Complete Ammonia Oxidizer

The complete ammonia oxidizer(comammox)is a microorganism that has both ammonia oxidation function and nitrite oxidation function,and can directly oxidize ammonia to nitrate.Traditional nitrification theory believes that nitrification is divided into two steps.The first step is to oxidize ammonia to nitrite by ammonia oxidizing bacteria(AOB)and archaea(AOA),and the second step is to oxidize nitrite to nitrate by nitrite oxidizing bacteria(NOB).The discovery of comammox breaks our perception of nitrification and is of great significance for improving the natural nitrogen cycle theory.The enrichment culture of comammox is the basis of its separation and identification,microbial mechanism analysis and engineering application.However,due to slow growth and difficulty in separating from traditional ammonia-oxidizing microorganisms,the enrichment and separation of comammox is very difficult.At present,there is only one pure strain and few enriched substances.It is of great significance to explore the influencing factors of comammox enrichment culture and accelerate its enrichment rate through optimization of various aspects.Enrichment of ammonia-oxidizing microorganisms is affected by many factors,including inoculum properties,reactor configuration,nitrogen source,and environmental factors.In this paper,the enrichment of comammox was the main research content.The optimization of inoculum,reactor configuration and nitrogen source type were used to accelerate the enrichment of comammox and obtain comammox enrichment.The main findings are as follows:1.Screened out the optimal inoculum by exploring the influence of water source type,water source geography and sample collection depth on the abundance of comammox and bacterial community structure in the surface water source sand filter.We collected 12 samples of sand filters,and compared the difference of comammox abundance and bacterial community structure in the river type water source and the lake type water source sand filters.The results showed that the water source type has a great impact on comammox abundance and bacterial community structure in sand filters.In sand filters of river-type water source plant,the abundance of comammox is generally higher than those of lakes,and it is significantly positivity correlated with the concentration of NH4+.The correlation coefficient is 0.826(p<0.01).The abundance of comammox in the five sand filters distributed from upstream to downstream of Qiantang River was analyzed.The relative abundance of Nitrospira in five sand filters was 0.1%,0.2.%,0.3%,13.2%,17.8%.And the abundance of comammox in the downstream water plant sand filter was higher than that of the upstream water plant.Comparing the difference of comammox abundance and bacterial community structure in different depths of sand filter,the results showed that there was obvious stratification of microbes in sand filter.In the depth of 30-40cm,the abundance and proportion of comammox was higher than the other 3 layers of samples.In summary,when select comammox enrichment inoculum from the sand filter,the water plant of the river type water source is superior to the water plant of the lake type,and the water plant downstream of the river is superior to the upstream,and in the same sand filter tank,the deeper layer sample is superior to the upper sample.Taking into account various indicators,the sand filter sample of 30-40cm in NX water plant is the best inoculum,which can be used for subsequent reactor enrichment culture.2.The effects of three reactor configurations on comammox enrichment and bacterial community structure were compared and the optimal reactor configuration was obtained.Three biofilm reactors were designed independently:sand filter reactor SC,packed ball reactor Q and soft filler reactor R.Comammox was enriched and cultured for 210 days using three reactors,and the enrichment efficiency of three reactors was compared.The reactor configuration has an effect on the nitrification activity of the microorganisms.The microbial nitrification activity in reactor Q is the highest,which is 0.94 mg N · g-1biofilm · d-1.The reactor configuration has a large effect on the enrichment efficiency of comammox.Reactor SC has the best enrichment efficiency on comammox,and the proportion of comammox increases from 0.47%to 10.19%with an increase of about 22 times.The reactor configuration also affects the community structure of ammonia-oxidizing microorganisms.The ammonia-oxidizing microorganisms in the SC reactor are always dominated by comammox,while in the Q reactor and the R reactor,AOB gradually wins the competition,and the abundance exceeds the comammox,becoming the domain microorganism for ammonia oxidation.Comprehensive consideration of microbial community structure and abundance of comammox,the sand filter reactor SC is the optimal reactor configuration for the enrichment of comammox in this study.3.The effects of three nitrogen sources such as nitrite,urea and ammonia on the enrichment efficiency and microbial community structure of comammox were explored,and the superior nitrogen source was obtained.Nitrogen sources have a certain effect on reactor nitrification activity,comammox ratio,and bacterial community structure.The nitrification activity of three reactors was significantly improved after cultivation,and the nitrification activity of the SB reactor with urea added and the SC reactor with ammonia were similar,which was higher than that of the SA reactor with nitrite added.The proportion of comammox in the SA,SB and SC reactors increased from 0.3%,0.4%and 0.5%to 4.5%,10.8%and 10.2%,respectively.The enrichment rate of urea and ammonia is similar,which supports the conclusion that comammox can use urea for growth and metabolism.The type of nitrogen source also has an effect on the bacterial community structure.The relative abundance of Nitrospira in the SA reactor with nitrite added is lower than that of the other two reactors.Considering changes in nitrification activity and comammox abundance,both urea and ammonia can be used as nitrogen sources for comammox culture,while nitrite is not suitable for comammox enrichment.