High Cell Density Cultivation Of Methanotrophic Bacteria

The application potential of Methanotrophs and methane monooxygenase is tremendous, as important multifunctional microbial catalyst, by numerous research institutions'attention. Give full play to the role of methanotrophs, methanotrophs not only can prevent and cure harm of greenhouse gases, still can obtain clean energy, plastic material, new food antioxidants, single cell protein etc., and indicates the huge potential in pollution control (such as inside trichloroethylene degradation). However, low growth rates and long fermentation cycle are the most important limiting factors for the application of methanotrophs. This thesis focus on the fast high-density cultivation of methanotrophs. The high-density system of methanotrophs can accelerate its applications in industrial biocatalysis and environmental methane control.Firstly we established the determination methods of cell dry weight, the specific growth rate, lag phase, and MMO activity.Then we investigates the cultivation of Methylosinus trichosporium 3011 on methanol and methane. It has been found that methanol although inhibits the growth of Methylosinus trichosporium 3011 above certain concentrations, is a good alternative due to its infinite solubility in water. Paraffin as the methane vector can also shorten the lag phase and help methanotrophs to reach high cell density. When the amount of copper ion,methanol and MV are 30μmol/L,0.05% and 2.5%(V/V) Methylosinus trichosporium 3011 has the optimum biomass and the highest MMO activity. The specific growth rate is 0.0460h-1, OD600 is 1.827, cell density is highest, cell dry weight can achieve 1.224gL-1, which were 6 times of the control, its lag phase shortened, activity has also improved. The separation and purification method of methanobactin (with Dianion HP20 adsorption,60% ethanol) from the fermentation medium has been established. The methanobactin(without Cu) purified from Methylosinus trichosporium 3011 has been added into the Methylosinus trichosporium IMV3011 Methylococcus capsulatus 3021,Methylosinus trichosporium 3011,Methylosinus trichosporium OB3b andGYJ3 with the concentration of 30μmol Cu2+ can affect the growth of IMV3011,IMV 3021,OB3b and GYJ3,such as:lag phase was shortened,the final concentration get higher,the specific growth rate become faster.,MMO activity has improved.