【作者】 郑璞；

【导师】 徐岩；

【作者基本信息】 江南大学 ， 发酵工程， 2013， 博士

【摘要】 丁二酸（琥珀酸）是一种重要的C4平台化合物，它作为有机化工的基本原材料广泛应用于食品医药表面活性剂清洁剂绿色溶剂生物可降解塑料等领域生物基丁二酸做为一种化工原材料的替代品，在石油基大宗化学品市场中日益受到重视，被美国能源部评估为前12种应该优先考虑的化学品之一秸秆是一种重要的木质纤维素类可再生资源，在我国资源非常丰富，每年产量约达7亿多吨，除少量被草食动物间接消化利用外，大部分以焚烧或微生物无用分解方式进入自然生态循环系统开展秸秆生物炼制丁二酸的研究，有利于减少传统化学合成中对化石原料的依赖，缓解大气的温室效应，促进碳氢化合物经济向碳水化合物经济转型；有利于促进秸秆的高值化利用，有利于解决―三农‖问题，增加农民收入本文围绕秸秆生物炼制丁二酸中的微生物菌株改造酶转化发酵策略和纤维素酶回用三个方面的关键问题，进行以下研究：(1)对丁二酸产生菌琥珀酸放线杆菌A. succinogenes CGMCC1593的pH温度和产酸三个表型进行改进首先，建立了基于―先选相对优势组群再选优势菌株‖策略的96孔板高效筛选法，通过选择性平板生长96孔培养板发酵HPLC浓缩检测和摇瓶复筛，实现了从大样本中高效筛选目标菌株其次，采用基因组改组方法，选育得到耐酸菌株F3-20和高产菌F3-3-f；采用温度驯化的方法，得到耐温菌株2-b F3-20在培养基pH5.6的条件下生长，菌体OD值提高了6倍，摇瓶产酸提高了32%；F3-3-f补料分批发酵，产丁二酸可达到94g/L，产率84%，生产强度1.96g/L/h；菌株2-b在40℃下分批发酵，产丁二酸可达到40.2g/L，产率89%，糖利用率92%，生产强度1.11g/L/h A. succinogenes的耐酸表型与丁二酸产量表型具有一定的相容性，但其温度耐受表型与产量表型的相容性相对较差再次，测定葡萄糖代谢关键酶活性发现：改组菌F3-3-f的EMP途径和C4途径中的部分酶（PEP羧化激酶己糖激酶1,6-二磷酸果糖醛缩酶和延胡羧酸酶）的活性增加，而C3途径中的部分酶（乙酸激酶丙酮酸激酶）的活性减少扩增片段长度多态性分析表明改组菌与出发菌在基因组上存有明显差异(2)探讨A. succinogenes发酵木糖与秸秆水解糖的特性以木糖混合糖和秸秆水解糖为碳源，摇瓶和发酵罐实验结果表明：A. succinogenes对木糖利用率高，但副产物乙酸浓度高，丁二酸产率低；混合糖或秸秆水解糖发酵时，其中的木糖和葡萄糖同时被利用，乙酸浓度高的程度得到一定缓解；以秸秆水解糖为底物，5L发酵罐中补料分批发酵48h产丁二酸77.6g/L乙酸9.4g/L糖利用率96%丁二酸产率84%生产强度1.62g/L/h比较单纯木糖和单纯葡萄糖发酵20h的菌体生长底物消耗和产物形成速率，认为木糖代谢过程乙酸浓度偏高可能与ATP需求有关，而丁二酸产率偏低可能与代谢过程还原力减少有关（3）系统研究A. succinogenes秸秆同步糖化（共）发酵（SSF）策略首先，考察β-葡萄糖苷酶（βG）和木聚糖酶对纤维素酶降解秸秆的协同作用，在纤维素酶中，按每克预处理秸秆（PCS）补充10CBU的βG或50-100U的木聚糖酶，秸秆水解率提高30%左右；摇瓶SSF产丁二酸浓度可提高2.5倍，得出：对于不同来源的工业纤维素酶可采用适当补充βG和木聚糖酶的方式进行秸秆水解或SSF其次，底物（PCS）浓度实验表明：合适的底物浓度为65-80g PCS/L，SSF最适底物浓度比秸秆水解的最适底物浓度略高再次，对SSF参数进行了系统表征SSF过程中，糖浓度维持在较低水平；纤维素酶活性与秸秆颗粒质量呈现相同的减少趋势，两者在发酵前期（12h内）快速衰减至一半左右，然后基本维持不变；而βG和木聚糖酶的活性基本维持不变；丁二酸与乙酸的质量比较低（4.5左右）进一步，通过采用预水解SSF工艺，有效提高了发酵过程菌体浓度，丁二酸浓度从45.4g/L提高到47.9g/L，产率从0.567g/g PCS提高到0.598g/g PCS，结合40℃发酵，丁二酸浓度达到52.4g/L，对秸秆产率为0.655g/g PCS，丁二酸/乙酸提高到5.6然后，比较分步水解发酵（SHF）和SSF的发酵参数及酶耗，得出两种发酵工艺路线各有利弊，总体差距不明显，采用自制T. reesei发酵液以及预水解同步糖化发酵工艺相对略好，但是酶的用量与价格是决定秸秆生物炼制丁二酸的主导因素（4）针对纤维素酶的回收利用，初步研究磁性固定化β-葡萄糖苷酶pH值敏感型可逆可溶性聚合物Eudragit L-100固定化纤维素酶的方法，以及固定化酶在水解秸秆中的应用首先，通过条件优化，制得磁性固定化βG，固定化酶活力和固定化酶收率分别达到6.67CBU/g与91.0%红外光谱和扫描电镜图证实壳聚糖Fe3O4和酶有机地结合在一起；Fe3O4除了提供磁性外，还促进了壳聚糖微球对酶的吸附与游离酶相比，固定化βG的最适pH向低pH偏移了0.3，表观米氏常数略有降低，说明固定化酶对底物的亲和性没有下降在协同纤维素酶秸秆水解体系中，固定化βG稳定性良好，反复使用8次（288h），平均秸秆水解率为76.9%其次，优化Eudragit L-100固定化纤维素酶条件，制得固定化酶的酶活力为5FPU/g，固定化酶收率16.2%；固定化酶的溶解pH范围比载体的溶解pH范围略向碱性偏移，酶反应最适pH也比游离酶的最适pH略向碱性偏移在秸秆水解体系中，其水解率仅比游离酶低3%，反复利用10次（400h），平均水解率为80.4%，没有明显下降的趋势当两种固定化酶同时用于秸秆水解体系时，水解时间略有延长，重复使用7次(420h），水解率仍有75%，展现了良好的操作稳定性和应用的前景更多还原

【Abstract】 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.更多还原