Cellulase Production Optimization In Trichoderma Viride R16 And Application In Lignocellulosic Lactic Acid Fermentation

Lignocellulose as the most renewable abundant and ubiquitous feedstocks in the world,has been widey utilized to produce high value-added biofuels and chemicals,such as alcohol,liquid fuels,acetic acid and lactic acid.Statistics show that there are tens of billions of ton lignocellulose in the world annually.Even in the domestic,there are more than eight hundred and forty million tons of straw and forestry waste available each year.In this master graduate dissertation,we choose Trichoderma viride R16 as starting strain,which undergo Co60 gamma-ray irradiation and screen from the mutant strain with high CMCase and genetic stability.T.viride R16 was studied optimal fermentation conditions for cellulase production,analysed cellulase enzyme components by protein mass spectrometry and researched the effects of inhibitors on the fermentation of cellulase.At last we explored the feasibility of the crude enzyme solution application on enzymatic hydrolysis of corncob,simultaneous saccharification and lactic acid fermentation.Research results are obtained as follows:In the shake-flask system,explicit the optimum medium and its fermentation condition of the cellulase fermentation,then we figured out the most suitable condition:4 g/L corn steep liquor powder,30%inoculum size,fermentation temperature 28℃,corn stover 30 g/L,fermentation time 120 h.As a result,the cellulase activity were increased 114.57%,rising from 0.51 FPU/m L to 1.09FPU/m L.And the results showing that notable increase of the cellulase activity was57.46%,45.99%and 21.52%at 20 g/L,25 g/L and 30 g/L corn stover content respectively after watering.Basised on these,we conducted a series of scale-up fermentaion using 5-L fermentator.The sample concentration of glucose,xylose and acetic acid,was measured by HPLC.The glucose was gradually depleted at about 24 h,and T.viride R16 were successively initiated the gene expression referring to hemicellulase and cellulase.Finally,we chose the condition of the washed corn stover fed-batch fermentation which was at 48 h add 30 g/L washed corn stover and 4 g/L dried corn steep liquor powder,and eventually cellulase activity was 1.85 FPU/m L and xylanase activity was 121.32 U/m L.It was produced from ligin degradation NH₃-H₂O₂ pretreatment that inhibitors such as ofp-hydroxybenzaldehyde,vanillin and syringaldehyde influenced cellulase fermentation.And the results showed that adding high concentration of vanillin and ofp-hydroxybenzaldehyde had an adverse effect on cellulase production and cell growth.Whereas,in the low concentration,it was not obvious.At 1 g/L syringaldehyde,T.viride R16 showed a high ability of degradation on syringaldehyde was 0.87 g/LMeanwhile,we conducted enzymatic hydrolysis experiment and simultaneous saccharification and lactic acid fermentation（SSF）for the compound enzyme solution.Enzymatic hydrolysis have shown that,8%corncob solid content,with adding100 m L 2.0 FPU/m L cellulase enzyme and 188.58 U/m L hemi-cellulase produced10.93 g/L glucose and 27.80 g/L xylose,0.48 g/g conversion rate of glucose from corncob were achieced.Under the same cellulase activity,we conduct simultaneous saccharification and lactic acid fermentation experiment produced 32.87 g/L lactic acid,and the conversion rate of lactic acid and acetic acid from corncob is 0.44 g/g.Proteomics analysis of cellulase solutions induced by NH₃-H₂O₂ pretreated corn stover exhibited a total of 681 various proteins and 2314 peptides were found in the fermentation broth.To estimate the percentage of each protein in the total identified proteome,raw-files were re-analysed using the i BAQ（intensity-based absolute quantification）method.And the cellulolytic enzymes,produced by T.viride R16comprise:0.74%EndogluanaseⅠ（EGⅠ）,13.77%Endogluanase III（EGⅢ）,6.75%EndogluanaseⅣ（EGⅣ）,2.39%EndogluanaseⅦ（EGⅦ）,7.96%ExoglucanaseⅡ（CBH II）and 0.92%endo-1,3（4）-β-glucanase（βG）and they all account for 40.06%of the i BAQ top 50 ranked protein mass.T.viride R16 also secreted three kinds of hemi-cellulase,swollenin,cellulose signaling related protein that auxiliary cellulose degradation and aromatic compound dioxygenase were also found which can degradate inhibitors.In the process of converting lignocellulose to lactic acid,the high enzyme liquid cost and the inhibition effect of inhibitors after pretreatment are the two major obstacles to the large-scale application of lignocellulose.In this study,we used the optimization strategy of fermentation technology and using cheap straw as the inducement material,to promote the mutant strain T.viride R16 strain produce the compound enzymes which can degradation of lignocellulose complex.Preliminary reduction of enzyme cost in lignocellulosic conversion process.