| Poly-γ-Glutamic Acid（γ-PGA）is an anionic biopolymer made up of D-glutamic acid and（or）L-glutamic acid throughα-amino andγ-carboxyl groups linked by amide bonds.The special structure ofγ-PGA gives it many excellent properties,such as biodegradability,water solubility and modifiability.Therefore,γ-PGA and its derivatives have great application potential in medicine,food,agriculture and environmental protection.At present,γ-PGA is mainly produced by microbial fermentation.The yield ofγ-PGA was up to30～50 g/L by screening the superior strain and optimizing the fermentation process.However,due to the polymer properties ofγ-PGA,the viscosity of the fermentation broth increases at the late stage of fermentation.The increase of the viscosity of the fermentation broth resulted in the restriction of the transfer of substrate,dissolved oxygen and heat,which affected the further increase ofγ-PGA yield.In addition,the high viscosity of fermentation broth makes the separation and purification process complicated and costly.Therefore,the problem of high viscosity of fermentation broth has become one of the key bottlenecks restricting the industrial scale production ofγ-PGA.Bacillus subtilis GXA-28 was aγ-PGA-producing strain screened from the natural environment in our laboratory at the early stage.The high viscosity of the fermentation broth in the late fermentation period limits the mixing and mass transfer of the fermentation broth,which is not conducive to bacterial growth andγ-PGA synthesis.In previous studies,KCl was found to have a significant regulating effect on the viscosity of fermentation broth.The high concentration of KCl（201.2 m M）in the medium will significantly reduce the viscosity of fermentation broth.In this study,B.subtilis GXA-28 was used as the research object,the law and mechanism of KCl regulating the viscosity change of fermentation broth was explored.The research was carried out from three aspects:fluidics,molecular structure and bacterial physiological structure.The effect of KCl（0 m M,67.1 m M and 201.2 m M）on theγ-PGA production was confirmed in the shake flask and fermentation tank.The results show that,the concentration of KCl in the medium increases from 0 m M to201.2 m M,the growth of the bacteria is inhibited,the yield ofγ-PGA is increased,the consumption of L-glutamate and glucose are accelerated,and the viscosity of the fermentation broth is reduced.The results confirmed that the addition of KCl could reduce the viscosity of fermentation broth,and the viscosity of the fermentation broth decreased with the increase of the concentration of KCl.In order to investigate the law of the viscosity change of fermentation broth regulating by KCl duringγ-PGA production,the fluidics of fermentation broth were analyzed.The high concentration of KCl（201.2 m M）in the medium will change the rheological characteristics of the fermentation broth.Consistency coefficient and yield stress were reduced by 58.96%and 32.50%,respectively,while the flow behavior index increased by 100%.The results showed that the addition of KCl enhances the fluidity of the fermentation broth,and the fluid properties of the fermentation broth are deflected to Newtonian.In addition,mass transfer characteristics and mixing characteristics have been improved to varying degrees.The volume oxygen transfer coefficient（k La）increased from7.96±0.29 h-1to 21.31±0.46 h-1,and the effective mixing volume ratio（T）increased from 16.71±0.15%to 25.13±0.35%.The results show that high concentration of KCl can reduce the viscosity of fermentation broth,resulting in corresponding changes in its rheological characteristics,mass transfer characteristics and mixing characteristics,which is beneficial to substrate consumption andγ-PGA synthesis.In order to explore the role ofγ-PGA molecular structure in the viscosity changes of the fermentation broth,the molecular structure（molecular weight,stereochemistry and conformation）ofγ-PGA synthesized under different KCl concentrations was analyzed.The high concentration of KCl（201.2 m M）in the medium will reduce the weight-average molecular weight ofγ-PGA by 13.93%.The proportion of D-glutamic acid was increased by 6.11%,static light scattering and shear viscosity measurement showed thatγ-PGA was easier to form aggregates in aqueous solution.Thermal analysis shows that the melting point,enthalpy change and thermal degradation temperature ofγ-PGA are increased by 1.4℃,11.5 J/g and 3.5℃,respectively.The infrared spectroscopy and circular dichroism analysis showed that the conformation ofγ-PGA changed fromβ-sheet to the coexistence ofβ-sheet andα-helix.The results showed that the molecular structure ofγ-PGA play an important role in reducing the viscosity of the fermentation broth.Early experiments have shown that high concentrations of KCl can inhibited the growth of bacteria.It is reported that the difference in osmotic pressure caused by salt concentration changes the permeability and fluidity of cell membrane.It is speculated that the physiological structure of the bacteria is related to the viscosity of fermentation broth.The high concentration of KCl（201.2 m M）in the medium will not change the morphology of the bacteria,but it will weaken the cross-linking between the bacteria andγ-PGA.The zeta potential of bacteria surface decreased from-70.49±3.35 m V to-81±2.46 m V,indicating that the cross-linking between bacteria and bacteria and the cross-linking between bacteria andγ-PGA was weakened.The integrity and permeability of the bacterial cell membrane were analyzed by propidium iodide and fluorescein diacetate,and the results shows that integrity of cell membrane decreased and permeability of cell membrane increased.Cell membrane fatty acid content analysis showed that the ratio of C12:0 fatty acids increased by12.36%,and the ratio of C16:0 fatty acids and C18:0 fatty acids decreased by6.83%and 5.64%,respectively.It is indicated that the fluidity of cell membranes was improved.The results showed that the physiological structure of bacteria play an important role in the process of KCl reducing the viscosity of fermentation broth. |
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