Isolation,Mutagenesis,and Fermentation Technology Of Lignin And Cellulose Degrading Microorganisms From Green Waste Decomposing

Recently,virescence business met a great progress in China,but also caused an increase of green waste yield.Biotreatment technologies like composting can realize the recycling of green waste,but the efficiency of which is limited due to the high content of lignocellulose.In order to accelerate the composting process,this study aims to isolate high-efficiency lignin and cellulose degrading microorganisms from green waste composting,the degradation ability of which was intended to increase through mutagenesis treatment,and study the gene function,fermentation technology and enzymatic properties of these microorganisms.Aniline-blue and carboxymethyl cellulose(CMC)-Congo red fade circle in medium,solid state fermentation test,and enzyme activity quantitative detection,were used to isolate the microorganisms.Three microorganisms with high-efficiency lignin and cellulose degrading were obtained.They were Bacillus subtilis(B.subtilis)BL03,Aspergillus sp.BL06,Geobacillus sp.BL15.B.subtilis BL03 can produces CMC-cellulase and laccase,Aspergillus sp.BL06 produces lignin peroxidase and manganese peroxidase,Geobacillus sp.BL15 produces CMC-cellulase.The atmospheric pressure room temperature plasma(ARTP)was used for mutagenesis of the isolated microorganisms.Dring the experiment,it was found Aspergillus sp.BL06 was not sensitive to ARTP mutagenesis for its mortality rate could not reach the requirement.No mutant strain with a significant positive mutation was found,after the first round of screening of Geobacillus sp.BL15.Therefore,the mutagenesis and screening of the two strains were suspended.The mutagenic conditions were controlled in order to induce mutation of B.subtilis BL03 with a lethality of about 95%.Based on the microplate reader,the first round of screening was carried out by using the decolorization rate of aniline-blue and combining with the size of the fading circle on CMC-Congo red medium.Then,two positive mutants were obtained after the second round of screening with the size of the transparent circle around the small filter paper and the quantitative detection of enzyme activity.And their gene stability was studied.A mutant strain B.subtilis BLAR1 was finally identified.After 20 subcultures,its CMC-cellulase and laccase activities were(U/mL):162.67 and 21.30,which were 49.69%and 35.18%higher than that of wild strain.B.subtilis BLAR1 and B.subtilis BL03 were applied to the green waste composting test,Phanerochaete chrysosporium(P.chrysosporium)and blank were used as controls.The composting agents were added twice,at the beginning of the experiment(0 d)and 30 d.It was found that:Firstly,the wild strain B.subtilis BL03 screened by CMC and aniline-blue had good lignin and cellulose degradation ability in green waste composting.After the 60 d composting,the degradation rates of lignin and cellulose were significantly increased than the blank control by 6.25%and 46.92%.Secondly,the mutant strain B.subtilis BLAR1 obtained by ARTP mutagenesis,significantly improved the cellulose degradation ability by 10.5%than B.subtilis BL03,but the lignin degradation ability was not improved.This result is far from the extent of the increase in enzyme activity in the laboratory culture state.A genome-wide analysis of B.subtilis BL03 and BLAR1 strains was studied by Illumina Hiseq Xten,and no significant difference was observed between the two strains in the GO functional gene pool.Analysis of genes in carbohydrate metabolism revealed that B.subtilis BLAR1 has increased one gene in the carbohydrate esterase gene than B.subtilis BL03,which is the EC 10 family gene in the CAZy gene database.The EC 10 family gene is associated with enzymes such as acetyl xylan esterase,cinnamoyl esterase,ferulic acid esterase,carboxylesterase,and S-formylglutathione hydrolase.It is speculated that the enhanced enzyme activity of the mutant strain is related to this and needs to be verified and further research.B.subtilis BLAR1 industrial production medium formula was obtained(g/L):glucose 9.0,beet molasses 15.0,corn syrup powder 20.0,soybean cake powder 20.0;optimum culture temperature was 37?,the economical shake speed is 200 rpm,the optimum initial growth pH range is 5.5-6.5,and the culture period is 24 h.A manual for the production of B.subtilis BLAR1 agent(see Appendix A)was presented,based on laboratory fermentation parameters and the experience of the agent factory.The maximum accumulation time of CMC-cellulase and laccase produced by B.subtilis BLAR1 was 48 h and 60 h,respectively;the optimum pH of enzyme activity was 6.0-9.0;the optimum temperature was 30-50?.These characteristics are beneficial to the enzyme system to play a good role in greening waste composting.In the enzyme inducer test,it was found that cellulose microcrystalline and corn cob significantly induced the CMC cellulase produced by B.subtilis BLAR1,while the induction of corn stalk and wheat bran was not significant.Guaiacol,vanillin and DL-phenylalanine have no significant effect on the laccase production of B.subtilis BLAR1.In view of the optimum growth temperature of B.subtilis BLAR1 is 37? and the optimum temperature of its CMC cellulase and laccase activity is 30-50?.In order to improve the effect of B.subtilis BLAR1 in green waste composting,it is recommended that:Composting should be carried out when the average temperature of the environment reaches 30?,and B.subtilis BLAR1 agent should be added.After being added to-the compost,the bacteria will rapidly multiply.After the composting temperature drops to around 40?,B.subtilis BLAR1 agent can be added again,to help the compost maturity.