The Production Of Methanobactin By Methane Oxidizing Bacteria-Photosynthetic Bacteria Co-Culture

Methanobactin(Mb)is a small molecular weight active fluorescent peptide secreted by methane oxidizing bacteria to the outside of the cell,which can be used as an important multifunctional biocatalyst.In this thesis,the co-culture of methane-oxidizing bacteria and photosynthetic bacteria was studied for the production of Mb.In the first part of this thesis,the determination method of methane-oxidizing bacteria and photosynthetic bacteria and the detection method of Mb were established.The chrome azurol S spectrop hotometry was used to measure the concentrations of Mb.The linear equation was y=-0.0475x+0.1228,which has a good linearity(R2=0.9963).The method has been used to detect the content of Mb.The growth of methane-oxidizing bacteria was characterized by the activity of methane monooxygenase(MMO).At the same time,the growth of photosynthetic bacteria was determined by modified spectrophotometry.The standard curve of photosynthetic bacteria was established at the concentration range of 1-5 mg/L.The linear equation is y=0.108x+0.26,which has a good linearity(R2=0.9976),Methane-oxidizing bacteria sole growth strain,photosynthetic bacteria sole growth strain,and methane-oxidizing bacteria&photosynthetic bacteria co-growth strain were obtained by controlling the light illumination,methane and carbon dioxide concentration.As for methane-oxidizing bacteria&photosynthetic bacteria co-growth strain,MMO activity of the culture obtained under light illumination was higher than that of the culture obtained under no light illumination.The results indicated that oxygen released by photosynthesis bacteria promoted the growth and metabolism of methane-oxidizing bacteria.Under the light illumination condition,Mb concentration was higher than no light illumination condition.This result proved that the culture of the mixed methane-oxidizing bacteria OB3b should be carried out under appropriate light illumination conditions.The second part of the thesis studied the effect of different light illumination conditions and initial carbon sources on the growth and Mb production of methane-oxidizing bacteria OB3b.The logistic growth kinetics model suitable for the growth of strain was used to obtain the lag phase,logarithmic growth phase and maximum specific growth rate of mixed bacteria growth.By comparison,the maximum specific growth rate of 0.0143 h-1 under light illumination conditions was obtained which was greater than that obtained under no light illumination conditions.The lag period was 18.74 h which less than 26.42 h.The results demonstrated that the light illumination was beneficial to the co-culture of mixed OB3b.When the light intensity was 30 ?mol/m2·s-1,the all light illumination time was 12 h,and the ratio of mixed gas CH4:O2:CO2(V/V)was 5:4:1,the maximum specific growth rate of the mixed bacteria OB3b was obtained and the lag period was the shortest.The mixed culture has highest Mb production.The results proved that the moderate growth of photosynthetic bacteria promoted the metabolism of methane-oxidizing bacteria and the production of Mb.The last part of the thesis discussed the effect of medium components on the growth and Mb production of mixed bacteria OB3b.Low concentration of copper ions promoted the secretion of Mb by methane-oxidizing bacteria,while excessive copper ions inhibited cell growth of mixed bacteria OB3b.Also,when the concentration of copper ions was high,the extracellular Mb secreted could form a Mb-Cu complex and return to the cells,and the concentration of Mb in the medium decreased.When the concentration of Fe3+was 5.0×10-4g/L,Mg2+was 0.15 g/L,NH4Cl was 0.5 g/L,H2PO4-was 0.3 g/L,the highest yield of Mb was obtained with concentration of 2.46 ×10-6 mol/L.