Optimization Of Culture Medium And Research On Cold Blast Drying Technology For Pseudomonas Putida

Bioremediation of oil pollution is one of the present research trend. Most of oil-degrading bacteria are gram-negative bacteria. However, gram-negative bacteria almost could not resist to the process of drying and preservation, which limits their commercialization and application. Pseudomonas sp., which belongs to gram-negative bacteria, has broad prospects in application of bioremediation, biotransformation and biological.In this paper, a pseudomonas putida, which can degrade crude oil, is the main subject. In this study, we first optimized the culture medium. Then we compared cold blast drying with spray drying to in order to an determine appropriate one, and optimized cold blast drying process to prepare high viable count bacteria powder. Finally we done some primary research about screening of collaborative strains and bioremediation of oily lake water under the situation oflaboratory. All the results are showed as below:1. We have completed the optimization of culture medium for Pseudomonas putida J7. We carried out the single factor and orthogonal experiments on choosing carbon source, nitrogen source and inorganic salts. The optimized culture media for strain J7 was as follows: glucose 6 g/L, yeast extract 12 g/L, Mg SO4×7H20 3.5 g/L, KH2PO4 2.5 g/L, Ca Cl2 1.5 g/L. Under the best condition, OD600 of bacteria got to 10.71 and the viable counts reached to 2.15×1010 CFU/m L, which was currently highest in China.2. We have done some research on the preparation process of Pseudomonas putida J7 bacteria powder by cold blast drying. First, we studied the effects of different ratio of carries to concentrate bacterium suspension on cold drying curve. Then we researched different factors on viability of cold blast drying and stability of preservation for bacteria powder, which was about the moisture content of bacteria powder, different carries, pre-treatment of carriers and protectives. The optimized cold blast drying preparation formular as follows: as bacterial cell carriers, diatomite and corncob powder pretreated by Ammonia were mixed with ratio1:2. The ratio ofcarries to concentrate bacterium suspension was 1:2. 7% mannitol, 5% sodium glutamate and 1% glycerin were mixed as protective agents(W/W). The moisture content was controlled less than 7%. In this condition, viable count was 1.03×1011 CFU/g in instant preparation, which was significantly superior to spray drying(24%). We concluded that cold blast drying was a suitable drying technology for Pseudomonas putida. The bacteria powder prepared by optimized cold blast drying method, had better preservation stability. After 150 d stroage at RT(room temperature), the viable count of optimized bacteria powder was 2.13 × 109 CFU/g. It was better than un-optimized bacteria powder(as control), which after 90 d storage at RT only remained 3.26 × 108 CFU/g. under 4 ℃ preservation conditions, optimized bacteria powder remained 3.36 × 1010 CFU/g after 150 d, which raised nearly double compared to un-optimized bacteria(1.84×1010 CFU/g, after 150 d storage).3. A primary study of the application of petroleum-degrading bacterium have been done. The collaboration strains of Pseudomonas putida J7 to degrade crude oil were Bacillus pumilus Y11 and Bacillus licheniformis B36. In the shake flasks bioremediation process of oily lake water, the oil degradation rate had an increase of 13.0% by mixed strains compared to a single strain. By adding organic nutrients into oily lake water, the oil degradation rate had an increase of 15% than adding inorganic nutrient. The study also showed that C/N of 20:1 was more appropriately for oil degradation. To imitate the oily lake water bioremediation in a static jar, the oil removal degrading rate increased to 56.6% from 20.1% by adding mixed strains and organic nutrients. The same as to the use of mixed oil-degrading bacteria powder, which remained considerable oil removal(54.7%).