Optimization Of Fermentation Conditions For ?-mannanase Production By Bacillus Licheniformis HDYM-04

?-mannanase is a hemicellulase that utilizes mannan as a substrate and widely distributes in plants,animals and microorganisms,and can be applied in many industrial fields.Bacillus licheniformis strain HDYM-04 was isolated in flax retting and demonstrated a high production of ?-mannanase.The process of enzyme production by strain HDYM-04 in 5 L fermentor has been farther studied in this thesis.The fermentation conditions in batch and fed-batch fermentation were optimized and the effects of controlled pH and DO values on production of ?-mannanase in batch fermentation were also studied.Besides,the batch fermentation kinetic models of HDYM-04 were established to make a foundation for the application of HDYM-04 in industry.The optimal fermentation conditions investigated by single factor method were obtained:the initial addition of konjac flour 2%(w/v),inoculum 6.7%(v/v)and the initial pH value of medium 8.0.The optimal process parameters including temperature,agitation speed and aeration rate optimized by orthogonal test were:temperature 37?,agitation speed 300 r/min and aeration rate 3 L/min.Meanwhile,an optimal time course of fermentation cycle was 30 h.Under the optimized conditions,the enzyme activity in batch fermentation could be up to 3298 U/mL,59%higher than that before optimization.The batch fermentation kinetics models of HDYM-04 were established and verified under optimal conditions.Three batch fermentation kinetic models were displayed as follows:Cell growth kinetic model:dX/dt=0.431X(1-X/15.522)Substrate depletion kinetic model:-dS/dt=1.11dX/dt+0.0002dP/dt+0.0008XProduction formation kinetic model:dP/dt=133.1dX/dt+222.87XThe correlation coefficients R2 of the three equations were 0.99021,0.98908 and 0.98812,respectively,which indicated good correlation between experimental values and models.Therefore,the equations could be used to describe cell growth,enzyme synthesis and substrate consumption process of HDYM-04 during batch fermentation.Subsequently,a further research was performed by controlling pH and DO value in batch fermentation based on the former optimal conditions.The two strategies were:1.when OD600nm?4.5,fix pH value at 7.0 till the end of fermentation;2.when OD600nm?4.5,fix DO%as 25%till the end of fermentation.The activity of the enzyme was not evidently increased,but the time of high level enzyme activity was greatly extended.Then the patterns of fed-batch fermentation were discussed.Results showed that:the pattern of adding 1%(w/v)konjac powder initially,then feeding 2%(w/v)konjac powder at late Logarithmic growth stage(OD600nm?4.5)was the best pattern for the enzyme production from HDYM-04 in fed-batch fermentation.The enzyme activity reached to 3374 U/mL,Finally,the maximum enzyme activity 3913U/mL was attained when adding 3%(OD600nm?4.5)konjac powder at late logarithmic growth stage(OD600nm?4.5),which was 89%higher than that before optimization,and the specific activity was 3411 U/mg as well.The fermentation conditions of HDYM-04 in 5 L fermenter were optimized in this thesis.Finally,the ?-mannanase activity from HDYM-04 could reach to 3913 U/mL which was 89%higher than that before optimization.Meanwhile the specific activity was 3411 U/mg.Besides,the establishment of batch fermentation kinetic models(cell growth kinetic model,substrate depletion kinetic model,production formation kinetic model)could make theoretical foundation and provide practical instruction for the application of HDYM-04 in industry.