Screening And Construction Of Compound Microbial System Of Lignin And Cellulose Degrading Bacteria From Slime Of Paper Mill

The suitable temperature,proper humidity and abundant nutrient sources of the paper machine environment provide perfect growth conditions for microorganism,which leads to the extremely high microbial diversity in the slime.Therefore,the slime of paper mill was selected as the source to obtained different species through the process of culture,isolation and purification.Then,species with efficient cellulose and lignin degrading were screened,and the characteristics of its enzyme production and degradation abilities were investigated.Last,the composite strains were constructed to improve the degradation characteristics of the screened strains and promote the resource utilization of agricultural waste.The main results are as follows:(1)The microorganism extracted from the slime of paper mill were obtained through the process of culture,isolation and purification,and the phylogenetic relations of different species were identified by 16 SrRNA technology.In this study,72 species were isolated and purified,belonging to 4 phyla and 16 genera.The dominant phylum,Proteobacteria,accounted for 75% of the isolated strains.The three most dominant genera were Pseudomonas,Acinetobacter and Bacillus,which accounted for 32%,20% and 14% of the total number of isolated strains respectively.(2)Through the qualitative experiment of Azure-B fade circle,ultraviolet spectrophotometer based quantitative experiment of Azure-B fade rates,Congo red/CMC-Na media,filter paper disintegration test,and the tests of enzyme activity of lignin and cellulose enzyme system,three efficient lignin degrading bacteria as well as three efficient cellulolytic bacteria were screened.According to the identification of phylogenetic relations,the lignin degrading bacteria were strain WH2-37(Pseudomonas sp.),strain BZ2-78(Paenibacillus sp.)and strain BZ2-84(Bacillus sp.).The three cellulose degrading bacteria were strain WH1-9(Bacillus sp.),strain WH2-56(Planomicrobium sp.),and strain BZ1-65(Xanthomonas sp.).(3)For the first time,in-depth study on the degradation capacity and enzyme production characteristics with different carbon source of Planomicrobium sp.WH2-56 was carried out.The maximum accumulation time of CMCase production of this strain was 48h-96 h,and the peak enzyme activity reached 0.62 U/mL.The optimum pH of CMCase production was 6.0 and the optimum temperature is 37?.When different cellulose materials and agricultural wastes were used as carbon sources,the enzyme production effect of the strain was different.Among them,microcrystalline cellulose and rice stalks had promoting effects on the enzyme production and metabolism of the strain.In these two carbon sources,the maximum enzyme activity of FPA was 0.35U/mL,and the maximum enzyme activity of CMCase was 0.62 U/mL,respectively.However,the enzyme activity and metabolic activity of the strain were lower when the corn straw was used as carbon source.(4)Three strains of high-efficiency cellulolytic bacterium and three strains of high-efficiency lignin degradation bacteria were compounded to no antagonism microbial system.The enzyme activity of CMCase and FPA,LiPs,MnPs and Lac were determined under continuous fermentation in 37?,200 rpm after 72 h,respectively.The enzyme activities were used as the targets for comparing to selected microbial system W-2,which with no antagonism affect to each other and with high lignin-cellulose degradation enzymes.In addition to the fact that the activity of lipase in this strain was not significantly increased compared with that of single strain,however,the activity of the other four enzymes had a larger or even doubled improvement compared with that of single strain.In particular,the activity of lignin enzyme laccase increased to 12.34 U/mL at most,which was four times as much as that of single strain.(5)Rice straw was used as the only carbon source in the media to determine the enzyme production characteristics of microbial system w-2.After adjusting the initial pH value and the culture temperature,the optimal conditions for the degradation of agricultural waste by the microbial system were determined.That is,when the culture temperature was 37? and the initial pH value was 6.5,the weight loss rate of straw reached around 32.15%,more than twice the rate of single strain WH2-56.It shows that the degradation capacity of microbial system w-2 is stable and reliable,and it has high practical application value and reference value.