| The development of alternative renewable energy has become a research hotspot,mainly because of the consumption of fossil fuels and the increase in environmental protection awareness.Butanol is considered to be a new type of biofuel with great potential due to its fuel performance and economic advantages.In recent years,the production of butanol through the fermentation of Clostridium acetobutylicum has received new attention.However,the high cost,low yield,and poor anti-butanol toxicity of bacterial cells of acetone-butanol-ethanol(ABE)fermentation are the main factors that limit the butanol production by C.acetobutylicum.Therefore,we have used carbon ion beam irradiation as a fast and efficient method to obtain C.acetobutylicum mutants,and studied mutation mechanism and practical fermentation of the excellent mutant.The following research was carried out:1.Carbon ion beam irradiation was used to mutate wild-type strain C.acetobutylicum ATCC824.After screening,the Y217 mutant with good yield,tolerance and genetic stability was finally obtained.C.acetobutylicum ATCC824 cells were irradiated with different doses of carbon ion beam by 80 MeV/u irradiation energy with the linear energy transfer(LET)of 40 keV/μm.The survival fraction of the strains after irradiation was explored by the plate colony counting method.Five mutants were obtained by gradient butanol-starch plates and fermentation rescreening.The five mutants have significantly enhanced butanol synthesis ability,and five consecutive generations to produce butanol relatively stable.The fermentation phenotype of the five mutants was studied by 72 h fermentation kinetic curve,combined with the cell growth research of the mutants under different concentrations of butanol stress and the stimulation of solvent,acid,and oxidative stress,and final selection to a higher production and tolerance of the mutant Y217.The stability test within 12 generations confirmed that it has good genetic stability.Butanol output stabilized at 13.67g/L.Subsequently,the response surface methodology(RSM)was used to verify the high butanol tolerance of Y217.We confirmed that the mutant can ferment to produce 8.35 g/L butanol after adding butanol,and the final butanol concentration in the fermentation broth reach 16.15 g/L.2.According to the aspects of cell surface morphology and characteristic,cell integrity and physiological and biochemical characteristics of cell membrane changes,explore the changes in cell surface properties and cell membrane physiological characteristics of Y217 under butanol stress.It can be observed by scanning electron microscopy that under the stress of 3.0%(v/v)butanol,the surface of ATCC824 cells appears wrinkled,dented and severely damaged,while the surface of Y217 cells is relatively smooth,typically rod-shaped,without obvious damage phenomenon,which is its enhanced tolerance preliminary evidence.In the hydrophobicity test,we found that Y217 under butanol stress reduces the cell surface hydrophobicity and decrease the entry of organic solvents into the cell,thereby improving the butanol resistance.In addition to hydrophobicity,the permeability of the cell membrane is also an important surface characteristics of the cell membrane.Through fluorescein diacetate(FDA)staining test,extracellular conductivity and intracellular macromolecule diffusion rate,we have fully proved that Y217 does not increase cell membrane permeability under butanol stress and maintains better cell integrity.In the study of the physiological characteristics of cell membranes,we focused on changes in cell membrane potential and membrane fatty acid composition.After Rh123 for cell staining,the changes in the membrane potential were estimated by the changes in fluorescence intensity.It was found that after 8 h of butanol stimulation,the membrane potential of strain ATCC824 decreased by 72.2%;the membrane potential of Y217 only decreased by1.7%,which can maintain high stability.In the study of fatty acids,it is found that the content of saturated fatty acids in the Y217 cell membrane has been greatly increased,which can increase the rigidity of the cell membrane to a certain extent,thereby reducing the fluidity of the cell membrane,which can reduce the entry of organic solvents into the cell and reduce the toxicity.Finally,the fermentation kinetics of the mutant Y217 and the wild-type strain ATCC824 were compared under the butanol stress by shaking flask fermentation,which verified the good fermentation performance of the Y217 mutant.3.Through the above research,we found that under the stimulation of butanol,the cell membrane of the Y217 mutant showed a big difference from the wild-type strain ATCC824.We predict that the high tolerance of Y217 is related to the modification of the cell membrane,so we conducted DNA-level mutation gene analysis.As expected,in the clusters of orthologous groups(COG)annotation classification of the Y217 mutant gene,it can be seen that the different gene functions are more annotated to the cell membrane-related physiological activities,which also reflects the metabolic and physiological bias of C.acetobutylicum mutant Y217 under the environment of butanol stimulation.Through screening,six important gene mutation sites encoding membrane-related functions were located in Y217,and the functions of these genes were analyzed in depth.The mutation of CAC 0033 and CAC0904 genes may change the permeability of the cell membrane by affecting the large-scale rigid structure rearrangement of the ATP-binding cassette(ABC)transporter.Mutations in the ptnd gene encoding the EIID of the phosphoenolpyruvate-carbohydrate phosphotransferase system(PTS)transport system Man II enzyme complex bound to the cell membrane in Y217 restrict the phosphorylation of carbohydrate molecules.By modifying the configuration of EIID,it may indirectly affect the characteristics of the cell membrane,leading to tolerance phenotype changes.Among the Y217 mutant genes involved in regulatory functions,CAC 0080 and CAC 3088 respectively encode components of the two-component system(TCS).Mutations in the histidine kinase Agr C(CAC 0080)will prevent the transfer of phosphate from histidine protein kinases to response regulators and hinder the adaptive response of bacteria to environmental stimuli.In addition,it is worth noting that there is an unreported predicted membrane protein(CAC 3309)that may be related to changes in Y217 cell membrane properties.4.Taking Y217 as the research object,conduct applied fermentation research.The content of related components in the corn powder medium,as well as the fermentation conditions were optimized by single factor preliminary study.The preliminary optimized corn powder,(NH4)2SO4,Ca CO3,K2HPO4,KH2PO4,Mg SO4 and Fe SO4.The optimal concentrations are 70g/L,3g/L,4g/L,1.5g/ L,1.5g/L,0.2g/L and 0.01g/L;the inoculum amount is 8%,the initial p H is 6.5-7.0,and the post-fermentation temperature is 34°C.According to this medium and culture conditions for fermentation,the final butanol yield reached 15.72g/L,which was significantly increased 15% compared to before optimization.Molasses is used as a carbon source for butanol fermentation,and its pretreatment methods and fermentation conditions are improved.The results show that molasses diluted to110g/L is more beneficial to the growth of bacterial cells;by comparing three different molasses pretreatment methods(filter,acid and heat treatment)it is found that acid pretreatment can decompose more sucrose in molasses into reducing sugars that are easily utilized by microorganisms to further promote the synthesis of butanol.In addition,the experimental results show that after 110g/L molasses(in P2 medium)is acid-pretreated,the exogenous addition of 15g/L glucose,2.5g/L peptone and1.0g/L neutral red can shorten the cell growth cycle and significantly improve butanol and total solvent production. |
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