| The preparation of nanocellulose nanocrystals(CNCs)materials from abundant and renewable natural lignocellulose is an effective strategy to realize the high-value utilization of lignocellulose.The preparation methods of nanocellulose include chemical method.physical mechanical method and biological method,in which,the advantages of biological method include mild reaction conditions,environmentally-friendly process,which make it has better market application prospects.So far,however,enzymatic preparations of CNCs were conducted in the low substrate concentration,and there are few reports on the preparation of CNCs using enzymatic methods under high solid loading.Based on the above background,to increase CNCs yield,in this paper,enzymatic preparation of CNCs at high solid loading was studied.Penicillium oxalicum strain was genetically modified to construct an engineering strain producing cellulase suitable for production of CNCs from eucalyptus dissolving pulp(EDP)at high solid concentration,and conditions of enzymatic hydrolysis were optimized.The comprehensive utilization strategy of hydrolysate and soild residue from enzymatic hydrolysis during the production of CNCs was also explored.This study provides a theoretical reference for the efficient transformation and comprehensive utilization of EDP.The main results of the thesis are as follows:1.Construction of engineering strain RE-6ΔC to produce CNCs from EDP at high solid loadingBy comparing the differences on production of CNCs from EDP through enzymatic hydrolysis with the cellulase systems secreted by lignocellulose degrading strains,it was indicated that the cellulase system from Penicillium oxalicum strain RE-6 had the best performance in preparing CNCs,but its high CBH(cellobiohydrolase)activity in extracellular cellulase was not conducive to the accumulation of nanocellulose.Therefore,the engineered strain RE-6ΔC was firstly constructed by knocking out the cellulase gene of CBHI(Cellobiohydrolase-I)in the strain RE-6.Compared with the original strain RE-6,it was found that in the enzyme compositions of RE-6ΔC,the specific enzyme activities of EG(endocellulase)and xylanase were increased by 34.1%and 47.4%,respectively,but the specific enzyme activitiy of exocellulase decreased by 48.4%,means that the enzyme system of the strain RE-6ΔC was more suitable than that of stain Re-6 for the preparation of CNC.Using the enzyme system from the engineering strain RE-6ΔC to hydrolyze EDP to produce CNCs at high solid concentration,it was found that,compared with the CNC prepared by the cellulase from original strain RE-6,the yield of CNCs was increased by 69.1%.The CNC prepared by the cellulase from the engineering strain RE-6ΔC was short filament-like with more uniform particle size,and had higher crystallinity and higher thermal stability and the CNC still maintains the type I cellulose crystal structure.2.Process optimization for the preparation of CNC by enzymatic hydrolysis with the cellulase from engineering strain RE-6ΔCIn order to obtain high yield CNC,the process conditions of CNC preparation by enzymatic hydrolysis with the cellulase system from the strain RE-6ΔC were optimized,including mechanical pretreatment,enzymatic hydrolysis conditions and hydrolysis process,and the effect of adding different EGs.It was shown that,using mechanical pretreatment to disperse pulp fiber for 5 minutes before enzymatic hydrolysis,then to wash with water,and centrifuged at 2400 g,the CNC could be effectively obtained.The optimum enzymatic hydrolysis conditions were that using 46 mg protein/g DM of cellulase to hydrolyze EDP for 50 h at 20%of solid concentration.Under the enzymatic hydrolysis conditions,the total CNC yield of 23.9%(based on initial weight of pulp)was obtained by a three-steps hydrolysis process,and after enzymatic hydrolysis,the final yield of glucose was 61.58%and the amount of residual solid substrate was 16.96%.Adding the endoglucanases rPoCel7B and rPoCel5B to the engineered enzyme system with a protein ratio of 1:1.06,the improvement for the CNC yield was the most significant(increased by 13.6%),and the CNC particle size was also reduced.It provides a reference for further modification of the engineering cellulase system.3.Comprehensive utilization of solid residues and hydrolysates from the enzymatic hydrolysis process during preparation of CNCIt was shown by the sugar components analysis that the hydrolysates contained high concentration of glucose and some oligosaccharides.Further hydrolyzing the hydrolystates with cellulase SCB18,the glucose concentration in the hydrolysates could reach 56.6 g/L,which could be converted into ethanol by yeast fermentation with high conversion efficiency(76.1%).It was found that the solid residues from the enzymatic preparation of CNC contained cellulose of 99.24%and carbohydrate of 101.2 w/%(which was calculated by cellulose),and the carbohydrate content meets the requirements of national standard of microcrystalline cellulose for food additives(GB 1886.103-2015).The polymerization degree of the solid residues was 121,which meets the quality standard of Chinese Pharmacopoeia(2020 edition).Therefore,the solid residues is expected to used as MCC in the food and pharmaceutical industries.It was also found by our research in this study that the solid resides can also instead of MCC to induce cellulase production during fermentation of filamentous fungi.The physicochemical properties of the residues were also analyzed and found that,compared with commercial MCC,the residues had lower crystallinity,smaller size.more uniform morphology,and better thermal stability,which means the residue performed better in the matrix materials and composite membranes.Through the construction of engineering strains,optimization of enzymatic hydrolysis process,and comprehensive utilization of hydrolysate and solid residue mentioned above,it is expected to obtain an enzymatic hydrolysis preparation process for nanocellulose at high solid concentration and achieve full component utilization of enzymatic hydrolysis solution and solid residue. |
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