| Organic solvent tolerant bacteria overcome the toxic effects of organic solvents with their unique morphology and structure,which enables them to grow and reproduce in organic solvents and produce a series of enzymes with organic solvent resistance,thus providing an effective source of enzymes for a wide range of non-aqueous enzymatic applications such as food,medicine,biology,and chemical industry.Aspergillus Niger C2J6 is a fungus independently screened by the project team to produce Organic solvent-tolerant Lipase(OTSL).In order to fully understand the characteristics of Organic Solvent tolerance of this strain,we synthesized organic solvents commonly used in food flavors and fragrants based on non-aqueous phase enzyme catalysis.A total of 35 kinds of organic solvents(alkanes,benzene rings,fatty acids,fatty alcohols,esters)were used to study the solvent tolerance of the strains.The correlation between the solvent and the toxicity of the strains was analyzed.The aim of this study is to provide an effective solvent reaction system for the subsequent whole-cell biocatalysis synthesis of OTSL and bacteria in the non-aqueous phase system.The specific research content and results are as follows:1,the tolerance rule of Aspergillus Niger C2J6 to organic solvents.Taking organic solvents as the carbon source of liquid fermentation,the growth tolerance of bacteria in organic solvents was investigated,and the tolerance rule of bacteria to organic solvents was grasps:(1)Aspergillus Niger C2J6 had a wide solvent tolerance.It could survive and grow in 20 organic solvents including 10 alkanes,3 benzene rings,5fatty alcohols and 2 esters,but no growth was observed in 9 fatty acid solvents.(2)In the survival experiment of organic solvent,after immersion in the environment containing only organic solvent(100%)for a period of time,the bacteria could still survive,but the immersion time of growth inhibition showed significant differences with different solvent types.The bacteria could still survive after 168 h immersion in liquid paraffin,but only 1 min immersion in n-butyl alcohol,isoamyl alcohol,and n-octanol.The bacteria are inactivated.(3)Through the impact growth experiment under the stress of organic solvents,it was found that the types and concentrations of solvents that the bacteria could tolerate were significantly different.The bacteria had the strongest tolerance to liquid paraffin and glycerin among alkanes and fatty alcohols,and the maximum tolerance concentration was 50%.Among benzene rings,the bacteria had the strongest tolerance to benzene,and the maximum tolerance concentration was 5%.Among the esters,the tolerance to methyl acetate and ethyl acetate was the strongest,and the highest tolerance concentration was4%.The bacteria could not tolerate fatty acid organic solvents.(4)Through the organic solvent adaptive growth experiment,it was found that when the bacteria were exposed to increasing concentrations of organic solvents,they could gradually adapt to higher concentrations of organic solvents and improve their tolerance.The growth rate of n-propanol and isopropanol was the highest(43%).2,Cytotoxicity of organic solvents against Aspergillus Niger C2J6.According to the 20 kinds of organic solvents in which the bacteria could grow,the toxicity of different kinds of organic solvents to the bacteria was investigated by taking the solvent hydrophobicity parameter log P as the measurement index.The results showed that:(1)The toxicity of organic solvents to the bacteria was correlated with log P,when0<log P<At 1.25,the solvent was the most toxic to the bacteria.1.25 & lt;log P<At 5.18,the toxicity of the solvent to the bacteria decreased with the increase of log P value,and there was a significant correlation between them.(2)The toxic effects of the bacteria to organic solvents were different at different growth stages: the cells in the delayed phase(12 h)and the rapid growth phase(24 h)were not sensitive to organic solvents and were relatively stable.Cells in the stable growth phase(48 h)and decay phase(72 h)were sensitive to organic solvents.(3)The concentration of organic solvents had a significant effect on the activity of glucose metabolism.The dose-response curve model of 20 organic solvents was established to quantitatively compare the toxicity of the solvents to the cells.Xylene >Cyclohexane >N-hexane>Isooctane >N-heptane >N-decane >Dodecanol >N-octane.3,Effect of organic solvents on the morphology of Aspergillus Niger C2J6 cells.Under the stress of organic solvents,the morphology of bacteria in liquid fermentation was significantly different with different solvents.(1)The mycelium pellets in the control group were round and smooth,and the mycelium morphology changed significantly under organic solvent stress,especially in n-propanol,methyl acetate,and ethyl acetate.The mycelium was white granular without spheroid.When the log P of organic solvent was 1.25-5.18,the dry weight and number of mycelium pellets increased with the increase of log P,but the diameter of mycelium pellets gradually decreased.(2)Light microscope observation of mycelium morphology under organic solvent showed that when 1% methyl acetate,benzene,cyclohexane,isooctane and liquid paraffin were added respectively,the tips of mycelium became thin and pointed,the branches decreased,and the cellular material in mycelium decreased.When 1% dichloromethane was added,the sporulation structure became deformed.When 1% n-propanol,methyl acetate and ethyl acetate were added,the sporulation structure did not form,but the tip became an enlarged globular structure,and the sporulation was deformed.(3)The characteristic morphology of cell surface observed by scanning electron microscope showed that the cell shrinkage under cyclohexane stress was the most serious,and the complete strip structure was almost no longer seen.Under the stresses of ethyl acetate,dodecanol,and n-octane,the mycelium pellet surface appeared obvious,collapsed,twisted,and adhesive substances.Under xylene stress,the cell surface was obviously swollen and its diameter was enlarged.(4)Transmission electron microscopy showed that organic solvents could destroy the cell structure,among which isoamyl alcohol was the most destructive to the structure of the cell.At 0.1%,the complete cell structure could not be observed,and at0.4%,the cell had changed from the original oval to irregular shape,the internal structure almost collapsed,the internal material was seriously leaked,and the cell growth stopped or even died.However,in the presence of n-hexane and n-heptane,the plasma membrane remained intact,transparent inclusions appeared inside,and mitochondria were still present in the cellular structure.In conclusion,the adaptation mechanism of A.Niger C2J6 to organic solvents was that the plasma membrane maintained the integrity of the cell,the mitochondria maintained cell metabolism,and the transparent inclusions appeared in the interior to prevent the damage of the cell structure by organic solvents. |
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