Screening Of Cold-Adapted Oligotrophic Bacteria And Research On Its Fermentation

An oligotrophic bacteria Ya was isolated from the water near the sewage outfall of South Lake. The result of the molecular identification of 16SrDNA showed that the homology between Ya and Bacillus subtilis is 100%, therefore Ya could be preliminarily determined to be a Bacillus subtilis. Using Ya and Yll which is a Bacillus pumilus preserved in the laboratory, we carry out an experiment of water purifying in normal temperature in comparison of 4 strains provided by Inner Mongolia Agricultural University, Y11 and Ya turned out to maintain relatively stronger ability in the degradation of organic matter, and CODMn is degraded from 16.34mg/L to 7.01mg/L and 7.69mg/L separately.By UV mutagenesis and screening in the condition of low temperature gradually, we lower the lowest temperature for growth of strains of Y11 and Ya from 20℃to10℃and 15℃separately.Compared these two hypothermia oligotrophic bacteria with 6 other hypothermia bacteria provide by Inner Mongolia Agricultural University, the results of water purifying in low temperature demonstrated that Yll maintained the strongest ability of water purifying in low temperature. In the condition of 15℃, CODMn can be degraded from 10.86mg/L to 7.73mg/L and 8.85mg/L by Yll and Ya. In consequence, we got a hypothermia oligotrophic bacterium.Based on the earlier study, Y11 has the ability to degrade phenol. Thence, it could be made into microbial agents for Phenol exceeding the micro-polluted water. Its in-depth study indicated that Y11 can tolerate the highest concentration of phenol was 400 mg/L. In artificial conditions, this strain can reduce 10mg/L phenol down to 0.094mg/L and 0.0068mg/L under artificial and natural water environments separately, which indicated Yll good low concentration of phenol degradation potential. Based on the study on temperature and pH, the optimum temperature for degradation of phenol is 28℃and optimum pH value is 8.0. As we know, oligotrophic bacteria were sensitive to heavy metal, so, we initiated a specific study on common metal ions, especially ions common in surface water. The result showed that Fe3+ can promote the degradation of phenol, while Mn2+(0.1mg/L-0.5mg/L), Cd2+(0.01 mg/L-0.1 mg/L) did not affect the degradation of phenol added. Additionally, Cu2+(0.1 mg/L-1.0 mg/ L) and Zn2+(0.5 mg/L-2 mg/L) also can inhibit the degradation a little bit. To sum up, Y11 can be used micro-polluted surface water, phenol treatment.Since mutagenesis by low-temperature oligotrophic bacteria Y11 showed good potential applications. Therefore, we initiated the optimization of its fermentation. Single factor analysis method had filtered out the best carbon source and nitrogen source, that was sucrose、corn flour、peptone, (NH4)2SO4、soybean meal, and also selected the best concentration of every factor. Then design response surface experiment on the basis of single factor analysis experiment,then ultimately got the best concentration of three significant factors that were sucrose 2.55%, peptone 3.41%, 0.32% soybean meal by BBD central composite experiment.Doing single factor screening experiment to optimum initial pH、fermentation time、VEM、incubation temperature on the basis of the result of RSM, eventually got the best fermentation culture medium and operating conditions, that were 2.55%(W/V)sucrose、0.32%(W/V)of soybean meal、3.41%(W/V)peptone,0.5%(W/V) (NH4) 2SO4,0.5% (W/V) of maize powder,0.04%(W/V) CaCl2,0.5%(W/V) NaCl,0.02% (W/V),MgSO4.7H2O; initial pH is 8,3% VEM, ferment--ation temperature is 28℃, Training time was 48h. Under these conditions the CFUs reached 230×108 cfu/ml,compared to the CFUs before optimized experiment which reached 40×108 cfu/ml, the CFUs increased by about 5.7 times.As the bacteria are oligotrophic bacteria, in the case of organic nutritional adequacy is difficult to produce spores. Therefore, production of the Bacillus conditions was studied. By identifying the best strain of Bacillus count the time and temperature of water bath 55℃,6 min. With distilled water,0.4% CaCl2 solution,0.3% CaCO3 solution,0.9% normal saline for treatment of the bacteria found,0.4% CaCl2 stimulated spores was the best, Bacillus rate of 94.96%.0.9% saline followed by the 89.43%.