Fed-batch Fermentation Technology Of Bacillus Thuringiensis BMB005

In this paper, fed-batch fermentation technology of Bacillus thuringiensis BMB005 was researched to enhance the titer of the culture. The impact of temperature, pH regulation, as well as the types, time, concentration and means of feeding carbon and nitrogen sources on yield of insecticidal crystal proteins (ICPs) and biological titer were investigated. By adopting the optimal technology, feeding 1% glucose in constant speed at both the latter exponential phase (the 10th ~ 12th h) and the fore-stationary phase (the 14th -16th h) in H9 medium at 30, the concentration of insecticidal crystal proteins and the titer increased 74.4% and 61.0% respectively to 7.85 mg/ml and 5403 IU/(j.l (Heliothis armigera to be tested).The results of fermentation temperature trials showed that the titer of the culture varied insignificantly when fermentation temperature were controlled at 28 , 30 and a altering temperature (30 for 16 h and then 28 ). The content of insecticidal crystal proteins increased 17.8% and the fermentation cycle lagged 5 hours when the fermentation temperature was done at 28 in contrast with 30. At the same time the content of insecticidal crystal proteins increased 10.5% and the fermentation cycle lagged 2 hours when the fermentation was done under the shirking tempreture (30癈 for 16 h and then 28).By feeding HC1 and NaOH solution to maintain the pH value of broth between 6.8 and 7.2 during the process of fermentation, the content of insecticidal crystal proteins and the titer increased 6.7% and 10.3% respectively. Then by controlling the pH value during the spore-forming phase and the spore-shedding phase, the yield of insecticidal crystal proteins and the titer increased 9.8% and 16.1%. The curve of pH showed that pH was not affected markedly during the lag and exponential phase by feeding l%o NFLiCl, though decreased a little during the mid-latter phase of fermentation. When l%o NtLjCl was fed in the medium, the yield of insecticidal crystal proteins and the titer increased 20% and 29% respectively. When 1% glucose was fed in constand speed at the stationary phase, higher yield content of insecticidal crystal proteins and titer were obtained than feeding l%o NHUCl.The results of experiments on choosing carbon resource showed that feeding glucose was most effective. When 1% glucose was fed in constand speed at the latter exponential and fore-stationary phase respectively, the titer increased 47% and 39%. The good result was owed to the increase of biomass when glucose was fed at the latter exponential phase, while the latter was owed to adaptive pH and more energy offered to synthesize product. Compared with feeding 1% glucose, feeding 2% glucose decreased the transformation efficiency of total sugar into products, and the phenomenon was observed by microscope that cell growth consistency decreased. As far as the feeding mode was concerned, constant-speed feed wasmore effective than batch-wise feed. The abnormal cellular form was often observed when glucose was batch-wise fed. Further experiment showed that better result was obtained when 1% glucose was fed constant-speedly at both the exponential phase (the 10th ~ 12th h) and the stationary phase (the 14th -16th h), and compared with the control, the yield of insecticidal crystal proteins and the titer increased 74.4% and 61.0% respectively; when 2% glucose was fed constant-speedly from the 10th h to the 18th h, the concentration of insecticidal crystal proteins increased 82.2% , while the titer increased only 49.0%.The concentration of insecticidal crystal proteins varied insignificantly when peptone was fed. In flask culture better result was obtained when amino acid was fed at the exponential phase than at the stationary phase. The yield of insecticidal crystal proteins increased 5.8% and 8.2% respectively when 0.15%o glutamate sodium and lysine were fed at the exponential phase. The yield of insecticidal crystal proteins increasing 42% was obtained when five different sorts of amino acid were fed simultaneously at the exponential phase. The res...