Study On Liquid Fermentation Process Of Nitrite-oxidizing Bacteria

Nitrite-oxidizing bacteria as one of two important physiological groups of nitrifying bacteria, had attracted more and more attention of researchers. Nitrite-oxidizing bacteria has the characteristics of slow growth rate and low natural conditions, which make the study of nitrite-oxidizing bacteria become more difficult and restrict its industrial production and application. Therefore, the research for growth rate and amount of the nitrite-oxidizing bacteria is extremely important.This subject for autotrophic nitrification bacteria y3-2(Nitrobacter sp.) which screened from laboratory, included experiments about the bacteria concentration and growth rate, and got the main research results as follows:(1) the feasibility of Real-time PCR for counting y3-2was established by comparing the counting results of Real-time PCR and MPN. Optimize The y3-2DNA extraction methods were optimized. Studies have shown that ultrasonic vibration could improve the efficiency of DNA extraction and when ultrasonic oscillation power was200W, the best ultrasonic shock time was2min.(2) The culture mediums were optimized by using single factor and orthogonal experiments, and the best medium formula of y3-2was CaCO30.5g/L, Na2CO31.0g/L, NaNO20.5g/L, MgSO4·7H2O0.12g/L, NaH2PO·2H2O1.16g/L, K2HPO4·3H2O0.33g/L, MnSO4·H2O0.0076g/L, ammonium molybdate50μg/L. Single factor experiments tested impacts of the fermentation conditions of inoculum size, temperature, pH and shaking speed to the y3-2. The best fermentation conditions:inoculum size10%, temperature28℃, initial pH of6.8～7.1before sterilization, shaking speed200rpm. After optimization, the Nitrobacter y3-2batch fermentation cycle was shortened by seven days prior to the optimization to four days. The bacteria concentration was improved from3.0×107cfu/ml to4.0×10cfu/ml. The bacteria concentration was improved two orders of magnitude.(3) The stability of the y3-2passage and tolerance to organic matter and sodium salt were investigated. Studies have shown that the y3-2passage was stable, and it was better tolerated of organic matter other than other nitrite-oxidizing bacteria reported in the literature. High concentrations of sodium salt could strongly inhibited y3-2. y3-2did not apply to seawater or other water which content of high sodium salt. (4) Batch fermentation and intermittent fill-batch fermentation of y3-2were studied, and50L fermentation was also experimented. The results of50L batch fermentation showed that the fermentation period was4d and bacteria concentration reached to5.0×109cfu/ml. The results of50L intermittent fill-batch fermentation showed that the fermentation period was4d and bacteria concentration reached to9.0×109cfu/ml. The experiments also determined that the concentration of supplemented NaNO2was0.3g/L, the start time of supplement was48h, and the end time of supplement was96h.(5) Four aspects of CaCCO3, dissolved oxygen, temperature, pH were studied to learn about the impact to the y3-2preservation. The best preservation was pH6.5, temperature-20℃,15%glycerol and sealing dark preserved. The most economical preservation was pH6.5, at room temperature and sealing dark preserved.