Screening And Identification Of Oligotrophic Bacillus And Its Fermentation Activity

Bacillus in oligotrophic is a kind of microorganism that can survive in a specific environment and be able to grow in oligotrophic condition.It can effectively reduce the ammonia nitrogen and organic matter in the micro-polluted water and improve the water environment.It has a good development prospect in the water application.In the study,the strain was firstly screened for growing well under oligotrophic condition which was preserved in the laboratory,the strain was identified based on morphological characteristics biochemical characteristics and 16S rDNA sequence and phylogenetic tree analysis.And secondly optimized the best fermentation conditions and culture medium for the strain.In the end,research under the condition of different cultivation methods on oligotrophic bacteria growth metabolism and the influence of dynamic fermentation,which can provide data support for the application of its mass.The main research contents and results of this study are as follows:?1?An oligotrophic strain K1 was screened from lab-preserved of Bacillus for identification and degradation of ammonia nitrogen,so as to develop an oligotrophic strain with characteristics of degradation of ammonia nitrogen in water.The strain K1 was identified as Bacillus cereus based on morphological characteristics biochemical characteristics and 16S rDNA sequence and phylogenetic tree analysis.The results showed that the maximum degradation rate of K1 on ammonia nitrogen and nitrate nitrogen was53.3%and 42.2%,respectively.?2?In order to increase the effective biomass and spore count of Bacillus cereus,the single factor screening,the results showed that the optimum fermentation conditions were as follows,fermentation of 24 to 48 h,culture temperature of 37?,initial pH value of 5.0,inoculation of 2%and the fluid volume of 50 mL/250mL.Adopting single factor method and Box-Behnken design were used to optimize the fermentation conditions,the optimal composition of fermentation medium was as follows?g/L?:glucose 30.25,peptone 12.40,MgSO₄ 0.81.On this condition,the biomass of bacteria was 1.7 times that ever before optimization,and the pH of fermentation broth was slightly higher than ever before optimization.?3?In this study,find out finally in oligotrophic conditions with adding different concentrations of glucose,NH₄Cl,KH₂PO₄,the results show that Adding different concentration glucose can promote cell growth,the higher the glucose concentration,the more obvious the promoting effect.However,in term of NH₄Cl,The higher the concentration,the stronger the inhibitory effect.In the end,adding KH₂PO₄ can significantly promote cell growth in fermentation prophase,but not in fermentation anaphase.?4?To study the effects of different culture methods on the growth and metabolism of bacteria,the results showed that cell density of bacteria in shaking table culture was 5 times higher than that in standing culture.Under shaking table culture,bacteria had relatively high utilization rate of phosphate and no significant influence on sugar consumption of bacteria.When dilution ratio was different,the effect of static and shaking table culture on the cell density of bacteria was research content.the results showed:When dilution was 5 times,the bacterial density in shaking table culture was significantly higher than that in standing culture.The dilution ratio continued to increase,the cell density was not significantly affected by different culture methods,and the pH of fermentation broth showed an upward trend.?5?The seed liquid obtained from standing and shaking table was transferred to culture under oligotrophic condition.the results showed as follows:The cell density and the utilization of protein consumption were significantly higher in the shaker culture than in the culture after transesterification.The utilization of total sugar in shaking table culture was slightly higher than that in standing culture.The activity of glycolytic enzymes?GK,PK,PFK and PDH?in shaker culture was significantly higher than that in shaker culture.The lower the concentration of nutrients,the lower the activity of glycolytic enzymes.