The Liquid Fermentation And Preservation Of An Ammonia-oxidizing Bacteria

Ammonia-oxidizing bacteria as one of two important physiological groups of chemoautotrophic nitrifying bacteria,had played an important role in the process about the conversion of ammonia to nitrite in nitrogen cycle.While the wastewater purification performed by microorganisms is increasingly known and have been widely applied by humans,the research and industrial productionof a novel nitrobacterial products have become a urgent task.However,the physiological characteristicsof strict chemoautotroph and slow growth making it more difficult to accomplish the high density cultivation of industrial production which has been restricting the development and application.In view of this,ammonia-oxidizing bacteria named AT7(（Nitrosomonas europaea）isolated from our laboratory was used for the study.In order to establish large-scale industrial production and preservation methods and achieve its industrialization,a series of exploration involved 4 aspects of media optimization、culture conditions optimization、fermentation mode and preservation methos had carried on.The main findings were as follows:（1）Experiment explored the impact of trace metals on AT7 growth.Studies had shown that as 0.031mg/L A and 0.031mg/L B added while in the presence of 6.04mg/L（Fe ion concentration）C,it can significantly improve the conversion efficiency of the ammonia.When the initial concentration of ammonia was 106 mg/L,the completion time of fermentation can be shorted from 7-8 days to 5-6 days after optimization,and the bacterial concentration increased from 3.7×10⁷ cfu/m L to 1.1×10⁸ cfu/m L;（2）The effect of adsorption culture on AT7 had been studied.Through experiments screened,the 200 mesh D was chosen as the best adsorbent material.With concentration of 1 g/L added,the complete conversion of 106mg/L ammonia only needed three days and no accumulation of NO₂-N in the process.And the bacterial concentration can bemaintained at least 1.0×10⁸cfu/m L after optimization;（3）The optimization about the buffer system of media、temperature、initial p H and shaking speed on the growth of AT7 was studied by using single factor experiments.The results of optimization were as follows:the optimal buffer was K₂HPO₄-KH₂PO₄of 0.02 M;the optimal temperature was 30℃;the optimal p H was 8.0-8.5;the optimal shaking speed was 200 r/min when the media volume was 150 m L in 500 m L flask;Via the media and culture conditions optimization,the best media obtained was Na Cl 0.3 g/L,Mg SO₄·7H₂O 0.03 g/L,C 0.0396 g/L,A 0.122 mg/L,B 0.078 mg/L,（NH₄）₂SO₄ 0.5 g/L,K₂HPO₄·3H₂O 4.108 g/L,KH₂PO₄ 0.272 g/L,200 mesh D 1 g/L;the optimal culture conditions was 30℃,p H8.0-8.5,shaking speed 200 r/min;（4）Established the feeding concentration of 0.4 g/L（NH₄）₂SO₄every time and feeding end time of 120 h by flask fed-batch fermentation experiments.Where fed once every 6 h during the day and 12 h at night,for a total feeding ammonia of 1023mg/L,the final bacterial concentration could be improved from 3.1-3.3×10⁷cfu/m L of bacth fermentation to 1.8-2.1×10⁸cfu/m L of fed-bactch fermentation after optimization;（5）Compared to the 50L batch fermentation,the period of fed-bacth fermentation was 5d and one day earlier to the before.The bacterial concentration could reach to 1.6×10⁸cfu/m L where improved 45.5%compared to batch fermentation when the fermentation ended;（6）Seven aspects of temperature,dissolved oxygen,p H,matrix substrate,antioxidants,inhibitors and carrier were studied to learn about the effect on the AT7preservation.The results showed that low temperature,low dissolved oxygen and ammonia andcarrier added was favorable to the preservation of AT7.The optimal preserved conditions was 4℃,in the dark,sealed and adding carried The bacterial activity retained 41.4%after 3 months;In summary,through this research,the concentration of bacteria and activity of nitrification had already reached advanced domestic level.It was fully equipped with the conditions of industrial production and can be adapted to most of the wastewater treatment applications.