Construction And Application Of Bacillus Licheniformis With Selective Degradation Of The Bamboo Component

At present,bamboo resources as a raw material of biomass were widely used in the area of natural bamboo fiber,pulping and paper-making.Bamboo was mainly composed of cellulose,hemicellulose and lignin,thus,the effective separation of various components of bamboo was the prerequisite to make the best use of bamboo.Microbial which existed in nature has poor degradation selectivity for lignocelluloses,in the process of 5 days fermentation,not only cellulose component of bamboo is degraded extensively,but also the high value-added compounds in the degradation products are usually absorbed by microorganisms quickly,thus,to a large extent,utilization efficiency of the resource is reduced,and it limits the comprehensive utilization of bamboo resources.In this paper,a bacterium which was screened out is turned out that it has very high capability of degrading bamboo,and it was identified to be Bacillus licheniformis20085,moreover,the enzymatic of degradation was systematically studied.Then,using molecular biology means to build a engineering bacteria of Bacillus licheniformis20085 which has ability of selective degradation of bamboo components.Optimize the technological conditions of bamboo fermentation,to realize Bacillus licheniformis fermentation bamboo pulp.The main contents of the paper are as follows:1.The degrading enzyme system of lignocellulosic which was secreted by Bacillus licheniformis20085 was explored systematically while bamboo powders are regarded as substrate.The enzymatic activity of cellulase degrading enzyme,hemicellulose degrading enzyme and lignin degrading enzyme were determined at a certain time interval;During 5 days fermentation,Fourier transform infrared spectroscopy(FTIR)was used to qualitatively analyze the changes of chemical components,meanwhile,quantitative analysis of chemical components in bamboo by chemical method of National Renewable Energy Laboratory(NREL).Enzyme activity assay showed that Bacillus licheniformis20085 has high activity of xylanase and lignin degrading enzyme activity,but the bacteria also has high cellulase activity;Component analysis showed that Bacillus licheniformis20085 could degrad cellulose,hemicellulose and lignin at the same time,the degradation rate of cellulose hemicellulose and lignin was 31.69%,26.85%and 27.11%,respectively.The results of the above experiments show that Bacillus licheniformis20085 has high degradation efficiency for lignocellulose,but the cellulose has a great loss during the degradation process,and it has poor selectivity of degradation.Thus,it can not be directly applied to the pulping process.2.At the same time,in order to solve the problem regarding poor selectivity degradation of Bacillus licheniformis20085,the genomic sequence of Bacillus licheniformis2008 was retrieved using CLC Sequence Viewer 6.8.All the endo-cellulase gene sequences were obtained by BLAST analysis and reported in the literature,the primers were designed according to the sequence of the reading frame.PCR technology was used to obtain a homologous arm sequence which is about 500bp in length.The homologous arm sequence was spliced with sequence of Cm~r by overlapping PCR method and then homologous recombination fragment is transformed into Bacillus licheniformis20085 competent cell to complete the knockout of the target gene,thus,we obtain two strains which are knocked out gene of cellulase,B.licheniformis20085Δcelb and B.licheniformis20085Δgh.The results of fermentation using B.licheniformis20085Δcelb and B.licheniformis20085Δgh released that the activity of endo-cellulase was reduced from 1.86 U/mL to 0.50 U/mL in B.licheniformis20085Δcelb,and the degradation rate of cellulose was significantly decreased,however,activity of laccase reaches to 1.42 U/mL,and it has no significant difference with the original strain.However,B.licheniformis20085Δgh has underperformed.3.Bamboo was fermented 5 days with B.licheniformis20085Δcelb and B.licheniformis20085Δgh.After 5 days fermentation,the chemical constituents of bamboo were quantitatively and qualitatively analyzed by means of weightlessness experiment,Fourier transform infrared spectroscopy(FTIR)and National Renewable Energy Experiment(NREL).The results showed that B.licheniformis20085Δcelb has excellent ability about selective degradation of lignocellulose,the loss of bamboo could reach to 17%or so;FTIR and NREL analysis showed that the degradation rate of the recombinant strain B.licheniformis20085Δcelb was only about 9.18%,while the degradation rate of hemicellulose and lignin retained 22.66%and 24.57%,respectively.And the bamboo was pretreated by chemical method,after process of pretreatment,fermentation which regards bamboo as the substrate with B.licheniformis20085Δcelb for 5 days to obtain yield,whiteness,Kappa number,viscosity of low-pollution bamboo pulp.In general,the above experimental results which proved that B.licheniformis20085Δcelb is an excellent strain of bamboo biological pulping.