Research On High-pressure Mutagenesis Breeding And Its Mechanism Of High-yield Bacterial Cellulose Producing Strain

Due to high crystallinity, high purity, good mechanical property, strong waterpermeability and water holding capacity, well biodegradability and synthesis of controlled,and nanometer size of thin fibrils (diameter0.01⁰.1um), bacterial cellulose (BC) was widelyused in the areas of food, medicine, organ reengineering, senior paper making and high-gradeacoustics etc. Because of the expanding application and the rapidly increased demand of BCin all walks of life in the future, how to increase the yield of BC comes to be a problemneeded to solve exigently. Optimizing culture medium, improving the conditions offermentation and cultivating high yield strains are the commonly used method of increasingBC yield. Cultivating high yield strains is solving the root of the high yield problem. Highpressure mutagenesis is one of the most effective ways. Among the methods of high-yield BCproducing strain mutation breeding, high hydrostatic pressure (HHP) technology bacomesone of the important ways of mutation breeding because of overcoming the shortages ofradioactive of traditional physical mutation (such as UV or X-ray, etc) and toxic of chemicalmutation (such as DES, nitrosoguanidine, lithium chloride and so on). Ultra high pressure (P≥100MPa), which can make changes in microbial cells form, cell membrane and cell wall,and cause intracellular biochemical reactions, even vary the microbial gene expression andthe nucleic acid structure and biological function, has great application potency in microbialmutation breeding.In the paper, a wild BC producing strain J2with good property was screened fromhomemade buckwheat vinegar in our laboratory firstly. Wild strain J2was treated by highhydrostatic pressure, then a mutant strain M₄₃₈with higher yield BC production and wellstablity in subculture was isolated successfully from all mutant strains. Both the fermentationmedium and the culture conditions of strain J2and strain M₄₃₈were optimized. Sequently, allindex parameters and properties of BC produced by strain J₂and strain M₄₃₈were measuredand compared. Next, the characterizations of morphology and physiology-biochemistry ofstrain J₂and strain M₄₃₈were researched and compared, and then the two strains wereidentified combining the phylogenetic analysis. At last, high pressure mutagenesismechanism of HHP technology on BC producing strain was primarily discussed in the level of DNA based on amplified fragment length polymorphism (AFLP). The main researchresults are as follows:(1) The wild BC producing strain J2with stable property was screened from homemadebuckwheat vinegar. Its seed culture medium, fermentation medium and culture conditionswere optimized. The optimum seed culture medium contained D-glucose7%, yeast extract1%, K₂HPO₄0.5%, MgSO₄·7H₂O1.5%, ethanol2%(v/v). The optimum cultivation time was24h at30℃with the shaking speed of150r/min in constant temperature shaking incubator.The optimized inoculation amount was7%. The optimum fermentation medium containedcarbon source3%(glucose:sucrose=1:2), yeast extract0.33%, FeSO₄0.4%, ZnSO₄0.09%,K2HPO₄0.1%,MgSO₄7H₂O1%, malic acid0.3%and anhydrous alcohol0.7%(v/v). Underthese conditions, BC yield could reach to10.41g/100mL, whch is1.22times as that under theconditions with no optimization (8.52g/100mL). The optimum fermentation time was7d, andthe yield of BC was about12g/100mL at this time.(2) After treating the wild BC producing strain J2by HHP, a mutated strain M₄₃₈withhigher yield of BC and stable property during serial passage was isolated from the mutants.The optimal conditions of strain J2mutated by HHP were under250MPa with15min at25℃.Srtain M₄₃₈cultured in the same seed culture medium and the same culture conditions withstrain J2's. The optimum cultivation time was24h at30℃with the shaking speed of150r/min in constant temperature shaking incubator. The optimized inoculation amount was9%.The optimum fermentation medium contained carbon source5%(glucose:sucrose=4:1),yeast leaching juice1.25%, CaCl20.15%, ZnSO₄0.2%, K2HPO₄0.2%,MgSO₄7H₂O0.93%,fumaric acid0.3%and anhydrous alcohol0.5%(v/v). Under these conditions, BC yield couldreach to28.99g/100mL, whch is1.84times as that under the conditions with no optimization(15.75g/100mL). The optimum fermentation time was7d, and the yield of BC was about34g/100mL at this time.(3) The main component of the gelatinous membrane produced by strain J2and strainM₄₃₈was confirmed as cellulose, and the content of it produced by the two strains were89.32%and89.35%, respectively. The other physical and chemical characters of BCproduced by strain J2were as follows: water content of wet BC was98.68%, rehydration rateof dry BC after absorbing water was81.76%, protein content of dry BC was7.69%, fatcontent of dry BC was1.65%, water holding capacity of wet BC was as94times as that ofdry BC, and water release rate of wet BC was56h. All above physical and chemicalcharacters of BC produced by strain M₄₃₈were98.73%,80.63%,7.83%,1.62%,105timesand80h, respectively. BC produced by strain J2and strain M₄₃₈was observed as dense mesh structure.The crystallinity indexes were78%and82%, respectively. The cellulose Iα contents were52% and62%, repectively. And the maximum tensile strength were55.8MPa and78.4MPa,repectively.The data above indicated that BC produced by strain M₄₃₈has more advantages thanthat produced by strain J₂. Not only the yield of BC of the mutated strain treated by HHP hasincreased, but the properties were enhanced.(4) Based on identification through phenotype and hereditism, strain J₂was varietas oftypical Gluconacetobacter hansenii strain, while strain M₄₃₈was subspecies of Gluconaceto-bacter hansenii. The components of optimum fermentation medium, culture conditions andthe BC yield of strain J₂and strain M₄₃₈were not the same, but both phenotypiccharacterization and phylogenetic relationships were completely the same. It confirmed thatHHP treatment could make changes in some biochemical processes in the bacteria metabolicpathway of BC producing strain, but it couldnot change the bacteria individual form, thegroup form, the fermentation state, the physiological and biochemical characteristics andthe genetic relationship.(5) Amplified fragment length polymorphism (AFLP) reaction systerm was constructed.It was feasible to conduct the AFLP analysis of Gluconacetobacter sp. and its mutated straintreated by HHP. The optimum technical parameters of the AFLP reaction systerm were asfollows:600ng genomic DNA served as template should be used in the digestion system withthe reaction time8hours, the time of ligation should be8h or overnight (more than10h),500times of dilution for the products of pre-amplification for selective amplification, and1pairsof primers (M+G/E+T) were selected which adapt the AFLP reaction system of Glucona-cetobacter sp. and its mutated strain treated by HHP. On the basis of AFLP analysis, strainM₄₃₈with higher BC yield was a deletion mutant induced by HHP, and the complement genesof the only deleted sequence code small multidrug resistance protein c100910, which inhibitBC producing in a certain extent. Therefore, the deletion mutant M₄₃₈muteted by HHP hasmuch higher BC yield than its initial strain J₂.