| Succinic acid (amber acid) is an important platform chemical for C4compounds whichwidely used in many fields including food, pharmaceutical, surfactants, detergents, greensolvents and biodegradable plastics. Bio-based succinic acid, as a substitute of chemicalmaterial, has been paid attention increasingly in the oil-based bulk and specialty chemicalsmarket. It identified by DOE as one of the priorities among the top12chemicals of greatestinterest. Corn straw is an important lignocellulosic renewable biomass and it is very abundantin China. The annual production amount of corn straw reaches7hundreds million tons, inwhich the majority is burn out or remains unused besides a small quantity for animal feedingand bedding indirectly. Therefore, biorefinery of corn straw for producing succinic acid isvery significance. Because it can not only alleviate the chemical industry on oil dependenceand the greenhouse effect, but also promote economy transition from hydrocarbons tocarbohydrate, increase high value-add of corn straw and income of peasants. Based on threemain aspects in bio-succinic acid production from corn straw, the key issues such asmicrobial strain improvement, strategy of enzymatic conversion and fermentation, andcellulase recycling, were studied in this work.(1) In order to improve acid-tolerance, thermo-tolerance and succinic acid production ofA. succinogenes CGMCC1593, a screening model named―selective plates-96-well platesfermentation-condensed HPLC assay-anaerobic bottles fermentation‘was developed andapplied in screening of the objected strain from a large-scale samples. Using genomeshuffling, an acid resistant strain F3-20and a high succinic acid-producing strain F3-3-f wereobtained. And a thermotolerance strain2-b was selected by domestication of increasingtemperature. As results, the growth OD of F3-20was six times higher than that of originalstrain CGMCC1593with succinic acid concentration improved32%over its original strainin anaerobic bottles fermentation. Under fet-batch fermentation of F3-3-f, the succinic acidconcentration, the productivity and yield were reached94g/L,1.96g/L/h and84%,respectively. And under batch fermentation of2-b at40℃, the succinic acid concentration,the glucose uptake rate, the yield and the productivity were40.2g/L,92%,89%and1.11g/L/h, respectively. Results suggested two phenotypes between acid resistant and high yieldwere compatibility at a certain extent, but two phenotypes between hot stress and high yieldhad less compatibility. Furthermore, comparing the fermentative parameters and the keyenzymatic activities of glucose metabolism among shuffled strains and its original, it werefound that the biomass of F3-3-f during fermentation was highest, as well as the activities ofsome enzymes in pathways of EMP and C4, such as PEPCK, Hexokinase, Fructose1,6-diphosphate aldolase, Fm were increased and in C3pathway, such as AK, PK were reduced. The amplified fragment length polymorphism (AFLP) analysis proved there wereexisted some genetic diversity among the shuffled strains F3-20, F3-3-f and their original.(2) Exploring fermentation characteristics of A. succinogenes on straw hydrolysate andxylose medium were performed. Different carbon sources including glucose, xylose, mixturesugar (of glucose and xylose) and straw hydrolysate were investigated under anaerobicbottles fermentation and fet-batch fermentation. Results showed the higher sugar utilizationrate, lower yield and higher concentration of by-products acetic acid in xylose fermentedliquid when compared with glucose fermented of F3-3-f. However, to a certain extent themixture sugar and straw hydrolysate fermented could alleviate the degrees of both yielddecline and acetic acid concentration increase. When straw hydrolysate as carbon resourceunder fed-batch fermentation in a5-L fermentor, F3-3-f produced77.6g/L succinic acid,9.4g/L acetic acid, and the sugar utilization rate, yield and productivity were96%,84%and1.62g/L/h, respectively. Comparing specific rates of cell growth, substrate consumption andproduce formation between glucose and xylose fermentation at20h, it might speculate thatthe higher acetate concentration and lower yield were correlated with ATP demand and thereducing power decrease in xylose metabolization.(3) SSF strategy of biorefinery succinic acid from corn straw was studied. Firstly thesynergistic role of β-glucosidase and xylanase in straw degradation by cellulase wereexamined. Results showed the enzymic conversion rate could be increased by30%when βGof10CBU/g PCS or xylanase of50-100U/g PCS was supplemented to the straw hydrolysisreaction, and succinic acid concentration of SSF under anaerobic bottles could be increased2.5times than the one that both βG and xylanase not added. A view of using cocktailcombination‘of βG and xylanase with various commercial cellulase was put forward inhydrolysis of straw or SSF. Secondly, by different substrate concentration tests, the optimalsubstrate concentration in SSF was found between65-80g/L, which was little higher than theconcentration of straw hydrolysis. Following the track of SSF, it was seen that the reducingsugar concentration maintained at low level. The cellulase activity as well as the weight ofPCS almost declined by half in early fermentation, and then maintained unchanged, whileboth activities of βG and xylanase were fairly constant. The ratio of succinic acid to aceticacid was4.3at fermentation end. An improved technics of pre-hydrolysis SSF was used, thesuccinic acid concentration was increased from45.4g/L to47.9g/L, and the yield increasedto0.598g/g PCS from0.567g/g PCS due to biomass enhanced during fermentation.Pre-hydrolysis SSF was performed at40℃by thermotolerance strain2-b,52.4g/L succinicacid concentration with a yield of0.655g/g PCS was achieved, and the ratio of succinic acidto acetic acid was increased to5.6. Finally, comparison fermentative parameters of SHF andSSF, it was showed that each of two fermentation process has advantages and disadvantages,although overall both difference is not obvious, the pre-hydrolysis SSF using T. reesei fermention broth as source of cellulase was slightly better relatively. However, we found thatenzyme dosages and price were the leading factors that biorefinery succinic acid of cornstraw depends upon.(4) For recycled usage of cellulase in hydrolysis of corn straw, immobilization ofβ-glucosidase on magnetic chitosan microsphere, immobilization of cellulase withsoluble-insolubl polymer Endragit L-100and their application in hydrolysis of straw weretaken initial attempts. Under optimized preparation condition, the immobilized enzymeactivity and yield of the obtained magnetic immobilized βG were6.7CBU/g and91.0%,respectively. The FTIR spectra and SEM of immobilized βG proved β-glucosidase wassuccessfully immobilized on magnetic chitosan microspheres which chitosan combined withFe3O4, Fe3O4also improved the adsorption capacity of chitosan microspheres expectproviding magnetic. The optimum pH of immobilized βG shifted to acid0.3and the apparentKm decreased comparing with native enzyme, which showed no change in the substrateaffinity between the immobilized and free βG. When the immobilized βG was applied inenzymatic hydrolysis of corn straw, the conversion rate was maintained above76%afterrecycling in8batches (total288h). The Immobilized cellulase activity and yield were5FPU/g and16.2%under the optimal condition by single factor experiments. The pH changerange of IC solubility slightly shifted to alkaline than the carried Eudragit L-100, and also theoptimum pH of IC was little higher than the free enzyme. In enzymatic hydrolysis of cornstraw, the conversion rate of IC was less3%than that of free enzyme, and there was75%after recycled7batches (420h) that showed good operational stability. Results exhibited theapplication potential of these two immobilized enzyme. |
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