Characteristic Study On Carbon Metabolism And Ammonia Metabolism Of Marine Ammonia Oxidizing Archaea(Nitrosopumilus Maritimus XMU1)

The whole denitrification process includes ammonia oxidation from ammonia to nitrite and/or nitrate,and the reduction from nitrite and/or nitrate to nitrogen gas.Ammonia oxidation,i.e.Ammoxidation,is the first and the limiting step in the whole process.Ammoxidation is mainly carried out by three categories of microbes,including ammonia oxidizing bacteria,ammonia oxidizing archaea and anaerobic ammonia oxidizing bacteria,called AOB,AOA and Anammox respectively.AOB was found more than one hundred years ago,while the first Anammox and first AOA was found in 1997 and 2005 respectively.Existing research shows that under the condition of high ammonia nitrogen,ammonia oxidation was usually controlld by AOB and Anammox.On the contrary,AOA prefers to oxidize ammonia to nitrite under low ammonia concentration?in nanomole concentration,like the equivalent of a tablespoon of ammonia in a large swimming pool?.Therefore,in the natural environment,such as oceans,AOA is considered the main contributor to ammoxidation in oligotrophic environments.However,molecular biology studies have indicated that the distribution of AOA is very wide.It is believed that AOA has many applications not only in the earth's ecosystem nitrogen cycle but also in advanced wastewater treatment and slightly polluted waters,etc.Until to present,there are only two pure AOA strains isolated due to the difficulty of screening and separation.Nitrosopumilus maritimus SCM1 is the only one AOA from marine.The cell of SCM1 is very small and culture solution of SCM1 looks like almost as clear as water after 15-days's culture.Cosequently,study on its physiological and biochemical characteristics is rather hard job.There seems no studies on SCM1 in China about its physiological and biochemical characteristics.Four years ago,our laboratory obtained SCM1 as a gift from University of Washington,focusing on exploration in advanced denitrification through high density cultivation.This paper is an important step for such a endeavor.And the following topics were carried out:First,a combination of CO₂ and organic compounds were useds as carbon sources?i.e.,mixotrophy?to study the characteristics of ammonia oxidation by SCM1.The results were compared with that of CO₂ as the sole carbon source?strictly autotrophy?to explore the effects of organic matter on ammonia oxidation.Second,under the condition of only organic compounds as carbon source,we studied the ammonia metabolism and cell proliferation of SCM1 to explore whether SCM1 can strictly heterotrophic growth and its ammonia metabolism.Third,under the different concentration of organic matter and ammonia nitrogen,the possibility of a mixed culture of SCM1 and AOB.After more than four years' cultivation of SCM1 in our laboratory,the results showed that the physiological and biochemical characteristics of SCM1 have obvious differences with previous literature reports.So we marked SCM1 in our laboratory as Nitrosopumilus maritimus XMU1.We have chosen the yeast extract,peptone,pyruvic acid and the main organic matters in the TCA cycle as carbon sources,carried out a series of ammonia oxidation experiments.Compared with the original culture medium?only CO₂ as a carbon source?,some carbon sources?pyruvate,citrate,fumarate?don't suppress XMU1 in low concentrations,but high concentration can inhibit ammonia oxidation of XMU1;Acetate and a-ketoglutarate have no inhibition or promotion of its ammonia oxidation;Yeast extract and peptone can promote ammonia oxidation of XMU1.These results are different from some results reported in the literatures.Under organic compounds as sole carbon source conditions,pyruvate,a-ketoglutarate could significantly promote the ammoxidation,whereas acetate have inhibition,citrate,fumarate,succinate at low concentrations play a role in promoting or not,but have inhibitory effect at high concentrations.The experimental results of pyruvate,a-ketoglutarate etc,as the sole carbon source show that XMU1 has heterotrophic growth metabolism.These results suggest XMU1 could grow in the environment that having rich organic matters and participate in ammonia oxidation.Therefore,we choose two typical AOB strains,Pseudomonas and Paracoccus,under high organic matter and ammonia nitrogen condition?10 mM acetate,5 mM ammonium chloride?,measured initial cell number and the final cell numbers of XMU1 and AOB,and ammonium and nitrite.Results show that XMU1 can reproduce under high organic matter and ammonia nitrogen,but its growth rate is lower than AOB,so AOB could take a succession of AO A during the long-term cultivation.Under XMU1+Paracoccus mixed culture condition,the fastest ammonia oxidation,and there is no nitrite accumulation,ammonium root thoroughly become nitrogen oxide.So we think there is collaborative relationship between AO A and AOB ammonia metabolism,but the specific mechanism needs further research.These results can help people to understand ammonia oxidation process of AOA,and also provide valuable research foundation for the subsequent ammonia oxidizing archaea species isolation,applications in advanced pollution water treatment and urban sewage treatment.