Occurance And Control Of Komagataeibacter Europaeus ACCC10220 Mutation

Inoculum of Ko,magataeibacter europaeus is generally prepared by static culture because of mutation occurrence induced by shear force under agitated conditions;meanwhile,bacterial cellulose(BC)pellicle in static culture is changed into spherical or star-like morphology by agitation.For the purposes of preparation of this bacterium inoculum with high biomass but without mutants and production of BC in the form of membrane using agitating culture,the following were studied in this dissertation:(A)the effects of types of culture vessel,medium,medium volume used,and culture time on mutant percentages in cultures;(B)comparison of mutants with wild-type strain in colonial morphology,products,physiology,DNA sequences of 16S RNA,rpoB,and key enzymes(including phosphoglucose mutase,UDPG-pyrophosphase,and cellulose synthase)involved in BC synthesis,RNA levels in genes for CMCase and phosphoglucose mutase,and patterns of intracellular total proteins;(C)effects of mesh installed in culture vessel and water-soluble polysaccharides added into medium on mutant percentages and BC structure.The results were showed below:(1)These factors,types of culture vessel,medium volume used,medium,and culture time,did influence mutant percentage.Two strains of mutant(M 1 and M 2)were isolated from agitated culture,while M 1 was presented usually in static culture.(2)There were no changes in physiology,DNA sequences of 16S RNA,rpoB,key enzymes involved in BC synthesis,and RNA levels in genes for CMCase and phosphoglucose mutase between mutants and wild-type strain,but their colonial morphologies and products were discriminated from each other and GroEL,a chaperone protein involved in proper folding and assembly of nascent proteins,was presented only in the wild strain.(3)Mutation did not occur when mesh(the height between the mesh and liquid/air interface ? 2 cm in static culture,and tightly installed along with inner wall of culture vessel in agitated culture)was used,or when water-soluble polysaccharides(agar 0.01%,or CMC-Na II 0.30%,or xanthan 0.30%)were added into media in static culture,suggesting that the mutation related with cell movement.Under agitated conditions,agar 0.10%,or CMC-Na ? 0.30%,or CMC-Na V 0.30%added into medium not only inhibited mutation,but also reduced crystallinity index and the degree of polymerization of BC.This result implied that mutation would be associated with BC structure,especially its the degree of polymerization.Based on these results,mutation of K.europaeus could be induced by BC stress,and not by shear stress commonly accepted.(4)Mechanisms of mutation induced by BC stress were not investigated in this dissertation,but agitated culture could be used to produce BC pellicle and prepare inoculum.Structure of BC pellicle(micro-morphology,crystallinity index and crystal sizes,the mass fraction of cellulose I?,and the degree of polymerization)obtained from the mesh in agitated culture was similar to those of BC in static culture.This paper first reported that K.europaeus inoculum with high biomass but without mutants and BC interference could be prepared by agitated culture.