Isolation And Identification Of A Cellulose-degrading Acinetobacter Pittii,Optimization Of Cellulose Degradation Conditions And Transcriptome Analysis

Termites use lignocellulose as a food,and the microbial flora in the intestine is rich in cellulase.Sodium carboxymethyl cellulose as sole carbon filter media was to screen and isolate the cellulose-degrading bacteria based on the local termite,Coptotermes formosanus,inhabiting the Liuzhou city,Guangxi Zhuang Autonomous Regin.The microorganisms capable of growing in the liquid and solid medium containing sodium Carboxy Methyl Cellulose as the only source of carbon were screened and isolated,namely BW.The results of the phylogenetic ananlysis of 16S rDNA gene fragments and the physiological and biochemical test show that BW was Acinetobacter pittii.The bacteria shaking culture of 20h showed the crude enzyme of the highest cellulose activity about 93.73±0.22U/mL with sodium carboxy methyl cellulose substrate when grown in 1%?w/v?sodium carboxy methyl ccellulose liquid medium and the culture biomass yield reached the maximum after 25h later at 37?,150r/min.The study of cellulose-decomposing bacteria of the genus Acinetobacter sp.is relatively rare,and BW identified Acinetobacter pittii as a novel strain resource for biodegradable cellulose.A growth Plackett-Burman design test matrix of Acinetobacter pittii was established to screen the T/?,pH,Mn²⁺/mM,PEG 8000/%,K₂HPO₄/%,?NH₄?₂SO₄?g/dL?of the OD₆₀₀ response.Zn²⁺/mM and peptone/%are important variables for cell growth,and their significance in cell growth is estimated.The model is used to screen the important factors affecting cell growth.The results of Plackett-Burman design test and fitting are determined by Minitab 18 software.The normal and variance analysis of the normalized effects of different factors on the indicators determined that pH,Mn²⁺,K₂HPO₄,?NH₄?₂SO₄,peptone and Zn²⁺were significant factors,and their effects on response values showed different correlations and significance.The degradation of sodium carboxy methyl ccellulose was optimized by single factor,Plackett-Burman design and Central Composite Design using Acinetobacter pittii.The pH,T/? and Mn²⁺/mM factors were determined by Plackett-Burman design to significantly reduce the ability of sodium carboxy methyl ccellulose to produce reducing sugar.The influence of the effect on the concentration of reducing sugar is in the order of pH>Mn²⁺>T/?,and the response of the regression equation by the Central Composite Design fitting the above three factors is the reducing sugar concentration??g/mL?=1408+607pH-253T+41.6Mn²⁺-45.5pH*pH+4.92T*T+0.138 Mn²⁺*Mn²⁺-1.5 pH*T+0.09 pH*Mn²⁺-0.07 T*Mn²⁺,predicted by Minitab software,verified by experiments,optimized predicted value and verified yield The higher the fitting value of the test value and the difference of R-sq?prediction?,the accuracy and credibility of the model is higher,and the optimized reducing sugar concentration is 4.28 folds of the previous fermentation medium.Through transcriptome analysis,differential gene analysis of Mn²⁺-treated Acinetobacter pittii and blank control,and online prediction of STRING protein interaction database and KEGG gene database,it is believed that Mn²⁺may promote the release of cellulase from Acinetobacter pittii,increasing the amount of enzymes of its cellulase,thereby increasing the cellulase enzyme activity.