| The complex and heterogeneous lignin is the most recalcitrant biopolymer in nature. It protects cell wall polysaccharides from microbial degradation. Thus, lignin biodegradation plays a central role in carbon cycle of terrestrial ecosystem. Additionally, the research of lignin biodegradation contributes to the development of lignocelluloses bioconversion technique and the application of bioremediation. The basidiomycetous white rot fungi are the most efficient degrader of lignin. Basic research on lignin degradation by white rot has been mainly carried out with Phanerochaete chrysosporium. It has been known that components involved in lignin degradation by P. chrysosporium contain ligninolytic enzymes, the low-molecular-weight glycoprotein, reductive factor and some small molecule substances. Actually, the mechanism of lignin biodegradation by P. chrysosporium remains to be in doubt.The thesis adopted two routes to study the mechanism of aspen cellulolytic enzyme lignin (CEL) degradation by P. chrysosporium. One was the construction of in vitro degradation system of aspen CEL with purified peroxidases. Another was the research of the extracellular active components produced by P. chrysosporium in aspen CEL degradation with a membrane-linked device that has patented:Firstly, the CEL was prepared from aspen, lignin peroxidase (LiP) and manganese peroxidase (MnP) were purified under the optimized condition of P. chrysosporium peroxidases production, and the carbon source of high co-production of by P. chrysosporium were investigated. Then In vitro degradation experiment of the aspen CEL with purified LiP, MnP, and pyranose 2-oxidase (P2O) was attempted. And using the optimal carbon source of co-production of extracellular ligninolytic enzymes and the optimized condition of peroxidases production by P. chrysosporium, the extracellular active components produced by P. chrysosporium during degrading the aspen CEL were studied with a membrane-linked device. Besides, for the unexpected finding in the experiment, the determination of alkali-soluble lignin concentration with Coomassie Brilliant Blue G-250 was studied. Finally, the total utilization of corn stalks with the selective degraded white rot fungi screened-Trametes hirsuta lg-9-was tried. The main results of this thesis are as follows:1. Preparation of aspen CEL and determination of alkali-soluble lignin concentration with Coomassie Brilliant Blue G-250The crude aspen CEL was obtained through crushing of wood chips, degreasing, grinding by ball-milling, enzymolysis by cellulase, extraction with 1,4-dioxane, reduced pressure concentration of extracted liquid, depositing with double distilled water and lyophilization. Further, the refined aspen CEL was obtained through the dissolution with 90% acetic acid, depositing the solution with double distilled water, vacuum drying, re-dissolution with the mixture of 1,2-dichloroethane and ethanol (v:v,2:1), depositing the solution with ethyl ether, washing with light petroleum and vacuum drying. Its residual sugar was 4.77%, its residual protein was 3.25%, its total lignin was 85.1%, its Mw was 15075 g/mol, and its Mn was 8595 g/mol.The preliminary investigation displayed that mixing Coomassie Brilliant Blue G-250 (CBBG) reagent and alkali-soluble aspen lignin or alkali-soluble alkali lignin resulted in the increase of the absorbance at the maximum wavelength 630 nm and mixing CBBG reagent and alkali-soluble sodium lignosulfonate resulted in the increase of the absorbance at the maximum wavelength 640 nm. The further research showed that the absorbance at the maximum wavelength of the mixtures was positively linear correlation with the concentration of alkali-soluble lignin. The isothermal titration calorimetric experiment and Fourier Transform Infrared (FTIR) spectroscopy comparative analysis of precipitates washed by water,4% ethanol, and 95% ethanol indicated that CBBG was non-covalently bound to alkali-soluble lignin. The estimation of lignin content in the aspen kraft pulping black liquor showed that the result of the CBBG method and Klason lignin were closer to quantitative preparation than UV spectroscopy. The CBBG method is reproducible, rapid, and cheap, and there is little or no inference from carbohydrate degradation products.2. Optimization of peroxidases production by Phanerochaete chrysosporium and purification of its peroxidasesThe study of the effect of the inoculation amount, culture temperature, concentration of veratryl alcohol on peroxidases production by P. chrysosporium showed that with the low concentration veratryl alcohol, the low inoculation amount favored to LiP and MnP production, while with the high concentration veratryl alcohol, the high inoculation amount favored to their production. P. chrysosporium produced the highest LiP activity on the sixth day at 30?, while the maximum LiP activity was obtained on the fourth day at 39?. As for MnP,30? was more beneficial to its high production than 39?. The critical veratryl alcohol oncentration of the highest LiP production was 3 mM. The effect of the concentration of veratryl alcohol on MnP production showed that the low concentration veratryl alcohol was in favor of its production at 30?, while the slightly high concentration veratryl alcohol was more benefical at 39?, but the too high concentration veratryl alcohol was adverse to the high production of MnP. In order to isolate and purify LiP and MnP, the spore suspension of A650nm=0.65/cm was used as inoculum, and static fermentation was performed with the addition of 3 mM veratryl alcohol at 39? of the short fermentation period. The extracellular liquid of 4 d fermentation was harvested. HI, H2, H3, H4, H6, H7, H8, H9, and H10 were purified by using ultrafiltration, gel filtration chromatography with Sephdex G-75 as fillers, ultrafiltration and dialysis, and Mono Q anion exchange chromatography. Wherein, H1, H2, H6, H7, H8, H9, and H10 all had LiP activity, and H2 had a trace of MnP activity. H3 and H4 have MnP activity, but with a trace of LiP.3. In vitro synergetic degradation of aspen CEL by peroxidases and P26Three systems were designed in the experiment that LiP composed of H6-H9, MnP composed of H3 and H4, and P2O from Irpex lacteus dft-1 degraded aspen CEL in cooperation. They were P system, C system, and V system, respectively. The P system contained 10 mg aspen CEL,3 IU LiP,4 IU MnP,0.3 IU P2O,0.15 IU inactivated catalase,4 ?m veratryl alcohol,5 ?mol Mn2+,500?mol lactic acid, and 500 ?mol glucose in 10 mL pH4.5 20 mM succinate buffer. The C system had the same components as the P system except that the inactivated catalase was replaced by the activated catalase. The V system had the same components as the C system except that 2 ?mol of vitamin C were added. The three systems were performed for 3 h,6 h,12 h, and 24 h, respectively. Enzyme assay, UV spectroscopy analysis of the supernatant, and GPC of the residual solid aspen CEL showed that with the increase of treatment time, there was an increasing trend for the content of dissolving lignin in the supernatant of the three systems. The inactivation of LiP caused by the excess H2O2 produced by P2O in the P system was slightly recovered by catalase in the C system, and further was recovered by vitamin C in the V system. The P system composed of peroxidases and P2O were able to efficiently degrade aspen CEL, catalase enhanced slightly the degradation of aspen CEL, and vitamin C further intensified the degradation.4. Influence of carbon source on the production of extracellular ligninolytic enzymes by Phanerochaete chrysosporiumThe effect of altering the carbon source in the growing environment was investigated relative to the production of ligninolytic enzymes by P. chrysosporium. Glucose, cellobiose, cellulose, and both mixtures thereof were used as the carbon sources, respectively. Monitoring the activity of ligninolytic enzymes during cultivation showed that glucose oxidase and glyoxal oxidase activities in all carbon sources were produced during cultivation. High peak levels (0.17-0.24 IU/mL) of MnP activity were observed only in mediums containing oligosaccharides. LiP activity was high in glucose medium (0.21 IU/mL of peak value); however, minimal amounts were formed in the cellulose medium (0.01 IU/mL of peak value). High amounts of cellobiose:quinone oxidoreductase (3.33-3.99 IU/mL of peak value) and cellobiose dehydrogenase (0.04-0.2 IU/mL of peak value) were measured when P. chrysosporium was grown on a medium containing cellulose. This work discovered that the mixture of glucose and cellulose as a carbon source more favored high co-production of ligninolytic enzymes by P. chrysosporium.5. Research of the extracellular active components produced by Phanerochaete chrysosporium in aspen CEL degradationThe membrane-linked device was used to study aspen CEL degradation by P. chrysosporium. P. chrysosporium was inoculated in Vessel A, and aspen CEL was placed in Vessel B. The device separated the mycelia of P. chrysosporium and the macromolecule lignin degraded in space, which can suppress interference that the mycelia absorbed lignin, but the membrane did not totally block the interaction between P. chrysosporium or its extracellular active secretion and the macromolecule lignin. The membrane-linked device that installed 1 kDa,5 kDa, and 10 kDa-membrane, respectively were used. The assay of the enzymatic activity and low molecular weight substances activity in the supernatant from Vessel A and Vessel B showed that the activity of LiP and MnP in the extracelluar enzyme secreted by P. chrysosporium in Vessel A can permeate into Vessel B through the membrane of 1k,5k, and 10k, however other ligninolytic enzymes and cellulases detected all cannot do it. The activity of reducing Fe3+and generating OH· were derived from P. chrysosporium''active secretion during cultivation, and the secretion whose molecular weight was lower than 1k has had the activity of reducing Fe3+ and generating OH·. The GPC of residual solid aspen CEL in Vessel B showed that the degradation degree of aspen CEL increased with the increase of the aperture of membrane installed in the membrane-linked device. Comparison and analysis of LC-MS/MS of the supernatant in Vessel A and Vessel B from the experiment group, control A, and control B showed that the P. chrysosporium in Vessel A from the experiment group produced some compounds, and these compounds produced some fragments that had the maximum molecular weight of 700-1000 in mass spectroscopy. Besides, those compounds neither were detected in the supernatant in Vessel A from control A, nor appeared in the supernatant in Vessel B from the experiment group. Therefore, the several kinds of compounds may be the critical components in aspen CEL degradation by P. chrysosporium.6. A mode of the total utilization of corn stalks using lignin degradation by white rot fungiT.. hirsuta lg-9 was the strongest fungus that selectively degraded corn stalk rinds (CSR) in the nine fungi. CSR was treated with T. hirsuta lg-9 and then refined into pulp. The biotreatment resulted in loss of paper strength and brightness, but energy consumption during refining (ECR) was reduced. Pearson correlation analysis between ECR and the enzymatic activity during biotreatment, the yield after biotreatment(Y), or the relative absorbance intensity on FT-IR of CSR after biotreatment showed that at a significant level of a=0.1, Y, the relative absorbance intensity at 3414 cm-1 (A3414), and the relative absorbance intensity at 1653 cm-1 (A1653) on the FT-IR of CSR after biotreatment had good linear correlations with the ECR. The ECR was positively proportional to Y and A3414, and negatively proportional to A1653. Further, multiple linear regression was carried out, for which ECR served as the dependent variable, and Y, A3414, or A1653 of the biotreated CSR served as independent variables. Results showed that the determining parameters of the biotreated CSR may be used to predict the ECR. In this work, delignified corn stalk pith (DCSP) was added to aspen alkaline hydrogen peroxide mechanical pulp (APMP). The DCSP enhanced the strength properties of the aspen APMP and inhibited yellowing. The biomechanical pulping of CSR has the potential to produce a low-cost green pulp, and the DCSP can serve as a pulp additive, realizing the total utilization of corn stalks. |
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