Mutation Breeding Of Bacterial Cellulose Producing Strains And Optimization Of The Fermentation Process

Bacterial Cellulose(BC)is a nano-scale polymer compound synthesized by bacteria and linked by?-1,4 glycosidic bonds with glucose-based,and has high purity.Bacterial cellulose has the characteristics of good water absorption,strong water retention,high tensile strength,good biocompatibility and biodegradability,and is widely used in food,medicine and chemical industries.At present,bacterial cellulose has been industrialized in Japan and the United States,and bacterial cellulose is a traditional food material in Southeast Asian countries.As a new type of nanomaterial,bacterial cellulose has great market prospects in China,but most of them are currently in the laboratory research stage,and there is little industrial production.This is mainly due to the lower yield of bacterial cellulose and higher costs.In order to meet the industrial production of bacterial cellulose,it is necessary to adopt measures such as selection of excellent strains,screening of fermentation medium,optimization of fermentation process to improve the fermentation yield of the bacterial cellulose.This article takes Komagataeibacter xylinus BCC529 which was the wild strain of bacterial cellulose as the research object.The high-yield strains were obtained by chemical mutagenesis of nitrosoguanidine(NTG)and primary screening and rescreening.The fermentation medium composition,culture conditions and fermenter process of high-yield strain and Gluconacetobacter xylinus P1 were optimized to increase the yield.At the same time,the bacterial cellulose produced by the three strains of the laboratory in static and agitated culture was studied in terms of yield,structural properties and application in beverages.The main results were as follows:1.Mutation and selection of bacterial cellulose producing strainUsing the BCC529 as the original strain,the optimized NTG mutagenesis method was as follows:the OD₆₀₀of suspension concentration was 0.4 of the original strain,the Tris-maleic acid buffer had a p H of 6.0,and the NTG mutagenized concentration was 0.05 mg/m L.After three rounds of mutagenesis,a high-yield strain SQ-N-20180711-2-6 was obtained from 745strains,and the BC yield reached 5.848±0.233 g/L,which was 118.37%higher than the original strain.The original strain BCC529 has 3275 coding genes with a total length of 3783578 bp and GC content of 62.14%.The high-yield strain SQ-N-20180711-2-6 was re-sequenced,the results showed that there were a total of 893 SNPs,of which 2 were in exonic,888 were in intergenic and 3 were in upstream and downstream;there were 22 Indel sites,1 in the exonic and 21 in intergenic.The COG annotation of the mutant gene in the exonic revealed that it was an Omp A Mot B domain protein involved in the biosynthesis of cell wall and membrane.2.Optimization of fermentation conditions for SQ-N-20180711-2-6 strain and P1 strainThe fermentation medium components and culture conditions of the high-yield strain SQ-N-20180711-2-6 and Gluconacetobacter xylinus P1 of the laboratory were optimized by single factor and response surface methods.The optimized fermentation medium for SQ-N-20180711-2-6 strain was:glucose 5.089%,corn steep liquor 2.02%,K₂HPO₄·3H₂O 0.3%,acetic acid 0.2%,Fe SO₄ 0.2%,and Mg SO₄·7H₂O 2%,p H 5.0,the BC yield was 7.145±0.158 g/L,which was38.94%higher than the initial medium.The addition of CMC-Na reduced the BC yield;adding0.3%oxygen carrier dodecane in the initial time,the BC yield reached 6.037±0.208 g/L,which was 11.65%higher than the control.The optimized fermentation medium for P1 strain was:sucrose 2.68%,corn steep liquor 1.63%,K₂HPO₄·3H₂O 0.3%,acetic acid 0.2%,Fe SO₄ 0.21%,CMC-Na 0.2%and Mg SO₄·7H₂O 2%,p H 5.0,the BC yield was 9.162±0.122 g/L,which was74.29%higher than the initial medium;adding 0.5%dodecane at 12 h of fermentation progress,and the BC yield can reach 4.106±0.207 g/L,which was 26.68%higher than the control.3.Optimization of fermenter process of high-yield strain SQ-N-20180711-2-6 and P1strainFermenter process optimization results of SQ-N-20180711-2-6 strain in 3L fermenter:When the bottom of the stirring paddle was six straight leaves and the middle was six oblique leaves(G4),the BC yield can reach up to 2.853±0.071 g/L,the group has the highest volume dissolved oxygen coefficient(KLa);the optimized fermentation medium in the fermenter had a BC yield of 4.094±0.202 g/L,which was 43.50%higher than the initial medium;when 0.3%oxygen carrier dodecane was added,the BC yield reached 4.697±0.231 g/L,which was 14.73%higher than the control.Fermenter process optimization results of P1 strain:When the concentration of CMC-Na was 0.2%,the yield can reach 4.435±0.203 g/L,which was 2.05times higher than the control;the BC yield of the optimized fermentation medium in the fermenter can reach 5.579±0.078 g/L,which was 25.79%higher than the initial culture;the BC yield of adding 0.5%dodecane in the fermenter reached 6.282±0.129 g/L,which was 12.60%higher than the control.4.Comparison of bacterial cellulose produced by different strains under static and agitated cultureThe bacterial cellulose produced by the Komagataeibacter xylinus BCC529,the Gluconacetobacter xylinus P1 and the model strain Acetobacter xylinum BCA263 under static and agitated culture was compared.The BC produced by the three strains under static culture was membranous,and the agitated were small clumps or fragments.After 96 h under static culture,the BC yields of BCA263,BCC529 and P1 were 3.972±0.045 g/L,2.481±0.182 g/L and 1.416±0.163 g/L,respectively.After 96 h under agitated culture,the BC yield was1.698±0.070 g/L,1.656±0.112 g/L and 1.715±0.087 g/L.From the production point of view,BCA263 strain and BCC529 strain were suitable for static culture,while P1 strain was suitable for agitated culture.Infrared spectroscopy showed that the hydrogen bond stretching vibration of BC produced by agitated culture was large;X-ray diffraction results showed that the BC crystallinity produced by static culture was significantly higher than that of agitated;the BC tensile strength produced by BCC529 strain under static culture was higher,P1 and BCA263were lower;Scanning electron microscopy showed that BC was a network structure,and it was denser under static culture.The diameter of the filaments of both culture cinditions was20?40nm;thermogravimetric analysis showed that the BC produced by static culture was more resistant to high temperature.The coffee milk beverage containing stabilizers BC under static and agitated cultured was observed for 24 h,and the stability was observed.It was found that the coffee and milk in the control without BC showed obvious stratification,while the BC added by agitated culture had no delamination.The stability of the suspension was higher than that of the control without BC.The precipitation rate measurement showed that BCA263 under agitated culture was significantly higher than that of static.The BC produced by agitated culture was more suitable as a stabilizer in beverages.