果实对酵母拮抗菌和外源水杨酸诱导的抗病性应答机理

用生物和非生物因子来进行采后病害的防治，是一个非常有效的方法。诱导抗性作为控制果蔬采后病害的生物技术，已成为该领域的一个研究热点。然而诱导抗性的机制非常复杂，涉及到寄主、病原菌、激发子之间的相互作用关系。本研究主要利用酵母拮抗菌Pichia membranefaciens和SA处理果实，观察其抗性诱导表达和对采后青霉病菌（Penicillium expansum）的抑制作用，并从蛋白质组学水平上对诱导抗性的机理进行了分析。研究结果表明： 1、酵母拮抗菌P. membranefaciens (5 × 107 cells·ml-1)和SA(0.5 mM)处理采后甜樱桃果实，能够明显地降低病害的发病率和病斑直径。酵母菌和SA处理影响到了果实抗氧化酶的活性，同时还改变了POD同工酶谱和甜樱桃果实的总蛋白含量，并诱导了新的蛋白质条带产生。用光学显微镜和扫描电子显微镜技术观察发现，在in vitro条件下P. membranefaciens能够紧密地结合与病原菌的菌丝，而在in vivo条件下这种结合较为松散。 2、借鉴其它模式植物的方法，我们建立了一整套适用于多汁类植物材料的蛋白质组学研究方法。对于芒果，桃，甜樱桃、苹果以及冬枣等果实，都取得了重复性非常好的2-D图谱。我们应用该技术进一步研究了P. membranefaciens (1 × 108 cells·ml-1)以及SA (0.5 mM)处理对桃果实蛋白质组的诱导影响。结果显示，两种激发子处理都能够诱导桃果实产生抗性，从而减轻青霉病引起的腐烂。在诱导处理1 d以后，酵母拮抗菌和SA分别诱导22和16个蛋白的差异表达。质谱鉴定的蛋白属于6大类：代谢，防御反应，转录，能量途径以及细胞结构。有6个蛋白受到两种激发子的共同调控。其中，4种蛋白(包括glutathione peroxidase, polyphenol oxidase precursor, catalase和methionine sulfoxide reductase) 属于抗氧化蛋白，涉及到活性氧代谢。另2个蛋白（Major allergen Pru av 1和peroxidase）是病程相关蛋白，直接参与植物的防御反应。同时一些磷酸化酶和转录因子也受到两种激发子的调节从而参与果实的抗病反应。酶学测定和Northern杂交的结果表明，拮抗菌与SA处理均能影响过氧化氢酶活性及其基因的表达。 3、采前用较高浓度SA (2 mM) 短时间（10s）处理不同成熟期的甜樱桃果实，能够明显降低果实青霉病的病斑直径，并能减轻较低成熟度果实的发病率。在没有接菌的情况下，SA诱导了33个差异表达的蛋白，其中用质谱鉴定出了26个。而在接种病原菌的情况下，SA诱导了19个差异表达的蛋白，并鉴定出了其中的12个。这些蛋白分别涉及到代谢、防御反应、转录、能量途径、信号转导等过程。在没有接种病原菌的情况下，SA处理诱导了Putative DnaJ heat shock protein, PR1-like protein, Peroxidase, Major allergen Pru av 1 (Pru a 1)和Catalase等与抗病有关的蛋白。而在接种病原菌的情况下，诱导了PR1-like protein, Peroxidase和Catalase蛋白的差异表达。通过酶活性测定以及对细胞学定位的研究，我们发现在没有接种病原菌的情况下，POD的活性受到SA的诱导。但是在接种病原菌以后，诱导效果不明显。

寄生植物在入侵植物防治中的作用——以两种菟丝子为例

生物入侵在全世界广泛发生，目前已经受到全球关注。入侵生物对群落生物多样性和生态系统功能造成严重威胁，导致严重的环境问题和惨重的经济损失。薇甘菊(Mikania micrantha)、五爪金龙(Ipomoea cairica)和南美蟛蜞菊(Wedelia trilobata)是我国华南地区危害最严重的三种外来入侵种，其中以薇甘菊危害最严重，是世界十大有害杂草之一。从20世纪80年代发生以来，薇甘菊已在我国广东农林区域造成严重危害。 机械防治、化学防治和传统的生物防治等治理措施，未能有效治理外来入侵种的危害，直到使用本地种菟丝子(Cuscuta spp)的防治策略。与从原产地引进有害生物天敌的传统生物防治方法不同，本地种由于适应当地气候且与其他物种协同进化，因此，对生态环境潜在的负作用小。从2000年，被发现寄生和抑制薇甘菊之后，菟丝子被认为是防治薇甘菊的有效措施。 为了探讨菟丝子寄生对外来入侵种的治理，及对入侵群落的恢复，本研究在内伶仃岛的林地(入侵种群落被引入菟丝子寄生1-4年)，以及东莞、深圳和海丰的干扰样地(入侵种群落被菟丝子自然寄生5年以上)开展野外调查。在每个样地分别设立外来种入侵亚群和菟丝子治理亚群，通过测定群落结构与组成、土壤性质与养分含量，以及外来种和菟丝子的生长与养分含量等参数之后，本研究得出以下主要结论。 (1) 虽然，被寄生的外来入侵种薇甘菊、五爪金龙和南美蟛蜞菊通过调节资源分配以抵御南方菟丝子(Cuscuta australis)的寄生影响，但是，菟丝子寄生导致外来入侵种生物量降低、繁殖能力下降、养分含量降低。虽然，很多寄生植物都是广谱寄生，能同时寄生多种寄主植物，但是，在本研究的被入侵的群落中，菟丝子主要寄生外来入侵植物。尤其是寄生于南美蟛蜞菊和薇甘菊的菟丝子，生长旺盛、繁殖能力强，表现出高度的适应性。因此，菟丝子对外来入侵种(南美蟛蜞菊和薇甘菊)有寄生偏好性，并对本地种的负面影响小。 (2) 通过吸收寄主的养分，田野菟丝子(Cuscuta campestris)有效地抑制了薇甘菊的危害。由于入侵种的凋落物养分含量高且分解效率高，而且，菟丝子能够促进其它凋落物的分解，并使难以被植物吸收的养分转化成易于被吸收利用的状态。因此，菟丝子与薇甘菊的寄生作用导致土壤养分含量的升高。在薇甘菊被抑制之后，本地种利用丰富的土壤养分资源，提高生长适应性，增强抵抗入侵的能力，甚至抵制薇甘菊的再生。 (3) 菟丝子的寄生作用改变了外来寄主与本地非寄主的竞争平衡，促进本地植物的生长与重建。在外来种被抑制之后，本地种的丰度和群落的物种多样性逐渐增加。本地种如：野葛(Pueraria lobata)和芦苇(Phragmites australis)，取代了入侵群落中的入侵种，成了群落的优势种。而其它原先被薇甘菊抑制的本地草本、藤本和灌木，在引入菟丝子防治之后长势较好。群落稳定性与物种多样性密切相关，被治理群落本地种的增加有利于群落的演替与稳定。 (4) 被干扰的生态系统往往更容易被外来种入侵，而外来入侵种又常导致人工干扰生境的严重退化。在人工干扰样地的菟丝子对薇甘菊的抑制效果与在林地的效果一致，导致被寄生的薇甘菊生长衰退、养分竞争能力下降、入侵危害能力降低。而在薇甘菊被菟丝子治理之后，土壤养分资源增加，入侵群落的物种丰度和生物多样性提高。本 地种的重建与本地群落的恢复密切相关，利于本地被治理群落的稳定，促进被干扰植被的恢复。 菟丝子是一种治理外来入侵种危害的有效措施，适用于破碎的生境和被干扰的生态系统，尤其是在采用目前防治措施难以治理的情况下。本研究为本地种防治外来入侵种提供科学依据，且表明以入侵地的本地种治理外来入侵种有可能成为有效且可持续发展的生物防治策略。

马铃薯Y病毒外壳蛋白基因的导入以及转基因马铃薯植株的大田攻毒

应用四种不同的马铃薯试管微型薯诱导体系生产试管微型薯，通过比较建立了—种有效的试管微型薯诱导系统。这种诱导系统使用液体培养基，具有试管微型薯发生频率高、薯块体积大和微型薯形成早的特点。同时，此系统使用的培养基成分成本低、方法简便以及所用设备简单，适用于容器内大批量生产马铃薯试管微型薯以及马铃薯种质资源的保存。 对银离子在马铃薯叶片组织培养过程中对愈伤组织诱导或芽分化和再生的影响作了研究。结果表明银离子通过抑制叶片组织培养过程中形成的乙烯与其受体的结合从而促进芽再生，但是其对叶片愈伤组织的诱导无显著效果。银离子的这些作用在通过同时应用2，4-D而明显表现出来。2，4-D通过促进乙烯的生物合成而降低银离子的促进作用，两者则通过对乙烯的调节而影响马铃薯叶片的愈伤组织诱导和芽分化再生。 将马铃薯Y病毒外壳蛋白基因通过根癌农杆菌双元载体系统导入马铃薯品种Desiree、K4和Favorita，获得了若干转基因株系。除了K4品种中—转化株系具有非正常生长形态外，其余转基因植株都生长发育正常。由此表明以根癌农杆菌介导的马铃薯转化中，构建于双元载体上的外源目的基因是随机进入并整合到受体细胞的染色体上。具有畸形生长性状的转基因植株的产生说明了PVY CP基因的整合可能干扰了控制正常生长发育、尤其是形态建成的基因表达。 在转化试验中，应用了试管微型薯薄片、茎切段和叶片三种外植体作为转化材料。对转化过程中农杆菌对外植体的侵染时间、共培养时间、外植体的类型以基因型对转化频率的影响作了比较研究。发现以试管微型薯薄片和茎切段作为受体的最佳侵染时间是十分钟，而叶片则为五分钟，三种外植体的最佳共培养时间皆为四天。在各种处理的最佳条件下，Desiree比K4具有相对较高的转化频率，表明马铃薯Desiree比K4在转化反应上更温和或顺从。 通过比较几种由不同统计得出的农杆菌介导的转化频率，认为使用“净转换频率”(Net Transformation Frequency)能更精确地表达马铃薯的转化效率。而在以前的报导中还没有—种统一的、并且能被广泛接受和使用的表达转化效率的参数或指标。． 以叶片作为起始材料的转化具有较高的转化频率。在转化外植体的植株再生过程中应用了2，4-D和AgN03两种乙烯调节剂分别于愈伤组织诱导和芽分化再生阶段，使其产生高频的植株再生。尤其是它的净化频率明显高于其它外植体的转化频率，并且无显著品种之间的差异，具有高效马铃薯转化系统的特征。 以聚合酶链式反应(PCR)检测转化再生植株得到的结果与DNA杂交(Southern blot analysis)的鉴定结果比较，结论是相同的。由此表明在以农杆菌介导的马铃薯转化试验中，PCR可被用于证实外源目的DNA的导入，它以简便、迅速的特点帮助节省时间以及提供及时的转化证据。 对三个马铃薯品种的一系列转基因株系在大田条件下进行了攻毒试验．最后从Favorita 品种中筛选出了两个抗性较强的无性系，它们具有明显较低的病毒侵染发生频率以及正常的生长发育性状，具有很大潜力成为生产上推广应用的抗病新品种。

细胞分裂素基因（T-cyt）对转基因植物生长发育的调控

高等植物基因表达过程中的信号传导是目前植物分子生物学研究的前沿和热点之一。不少研究者已将脱落酸、乙烯、细胞分裂素及其它植物激素的作用一起归之于植物基因表达的信号传导系统。细胞分裂素作为—类重要的植物激素在植物的生长和发育过程中起重要的调节控制作用。因此研究细胞分裂素的基因与植物发育过程的关系是十分重要的。 为研究细胞分裂素对植物基因表达的调节，本文从转录和翻译水平上测定了黄瓜子叶在外源细胞分裂素诱导下微管蛋白基因表达的活性。发现经BAP处理的黄瓜子叶中α,β-tubulin mRNA迅速积累，微管蛋白的含量迅速增加。这表明外源细胞分裂素在诱导黄瓜子叶膨大的过程中激活了微管蛋白基因的表达。 为探索不同启动子驱动下的细胞分裂素基因转入植物后的表达对转基因植物生长发育的调控，本文将来自根癌农杆菌的细胞分裂素基因(T-cyt)分别置于CaMV 35S启动子，rbc S启动子和T-cyt基因自身启动子的调控下，构建了嵌合表达质粒，分别转化烟草和马铃薯。转基因烟草和马铃薯的PCR检测和Southern杂交鉴定均证实T-cyt基因已分别整合进烟草和马铃薯的核基因组中。标志基因NPTⅡ的酶活性测定表明有外源基因的表达。转基因烟草的Northern分析表明：CaMV 35s启动子驱动的T-cyt基因的mRNA在叶、茎和根中均有表达;rbc S启动子指导的T-cyt基因在叶中表达最强，茎中较弱，在根中几乎没有表达。转细胞分裂素基因的烟草在生长发育上与未转化的对照相比有明显不同。转基因烟草中叶绿素a，b含量明显增加，叶面积减小，叶衰老迟缓。T-cyt基因转化的烟草顶端优势受到抑制，侧芽生长旺盛;与对照相比，其节间短，株高降低，根生长受抑制。 本文还构建了T-cyt基因自身启动子与报告基因GUS编码区的嵌合表达质粒，转化烟草和马铃薯以研究T-cyt启动子在植物中的表达。组织化学定位测定表明，T-cyt启动子在植物的茎，叶中的表达较强，特别是在腋芽的生长点有很高的表达活性，但在根中的表达较弱。诱导性表达试验表明，T-cyt启动子的表达强度受细胞分裂素的诱导，而生长素对T-cyt启动子的表达无明显影响。这提示T-cyt启动子是一个细胞分裂素诱导性表达的启动子。 总之，将T-cyt基因转入植物，作为调节内源细胞分裂素的一种手段，可以对植物的生长发育进行调控。尤其是利用发育阶段特异性和各种器官特异性表达的启动子可以调节T-cyt基因的表达活性，有可能创造出具有经济价值的、具有新遗传特性的植物。

Utilization of several plant proteins by gibel carp (Carassius auratus gibelio)

Six isonitrogenous (gross protein content 35%) and isoenergetic (gross energy content 17 kJ g(-1)) diets were formulated to investigate the effects of inclusion of plant proteins on the gibel carp (Carassius auratus gibelio L.). The plant proteins tested were: soybean cake (SBC), potato protein concentrate (PPC), peanut cake (PNC), cottonseed cake (CSC) and rapeseed cake (RSC). Fish meal (FM) was used as control. In each diet, 27% of the protein was supplied by fish meal, and the rest supplied by the plant protein tested. Each diet was fed to three groups of gibel carp for 8 weeks in a recirculation system. Specific growth rate (SGR) in fish fed the control diet was significantly higher than those in the other groups, and SGR in fish fed the PPC was significantly lower than in fish fed other plant proteins. There was no significant difference in SGR among the other groups. Feeding rates were ranked in the order: RSC > CSC > FM > PNC > SBC > PPC. Conversion efficiency was highest in groups fed FM, SBC and PNC, followed by groups fed CSC and RSC, and was lowest in the group fed PPC. The fish fed PPC showed lower protein retention than those fed FM and SBC. FM showed highest energy retention while PPC showed lowest, There was no significant relationship between SGR and intake of digestible protein (g g(-1) day(-1)), digestible lysine (g g(-1) day(-1)), digestible methionine (g g(-1) day(-1)) or digestible total essential amino acids (g g(-1) day(-1)), suggesting that the differences in SGR could not alone account for any of these variables.

Hydrogen production characteristics of the organic fraction of municipal solid wastes by anaerobic mixed culture fermentation

The hydrogen production from the organic fraction of municipal solid waste (OFMSW) by anaerobic mixed culture fermentation was investigated using batch experiments at 37 degrees C. Seven varieties of typical individual components of OFMSW including rice, potato, lettuce, lean meat, oil, fat and banyan leaves were selected to estimate the hydrogen production potential. Experimental results showed that the boiling treated anaerobic sludge was effective mixed inoculum for fermentative hydrogen production from OFMSW. Mechanism of fermentative hydrogen production indicates that, among the OFMSW, carbohydrates is the most optimal substrate for fermentative hydrogen production compared with proteins, lipids and lignocelluloses. This conclusion was also substantiated by experimental results of this study. The hydrogen production potentials of rice, potato and lettuce were 134 mL/g-VS, 106 mL/g-VS, and 50 mL/g-VS respectively. The hydrogen percentages of the total gas produced from rice, potato and lettuce were 57-70%, 41-55% and 37-67%. 2008 International Association for Hydrogen Energy.

宁南山区施肥对马铃薯生长发育、产量及品质的影响

为探讨在宁南山区不同施肥处理下马铃薯生长发育和产量最佳施肥方式,采取随机区组设计进行田间试验,开展对马铃薯产量和品质影响的研究。结果表明,在当地条件下,处理10收获期干物质积累总量平均比对照增加30.97%,各施肥处理收获期的干物质累积量比对照增加7.35%~73.31%。马铃薯生长发育最佳施肥方式是N 300 kg/hm2、P2O5200 kg/hm2、K2O 200 kg/hm2、M 17.5 t/hm2,(氮肥基施和追肥各一半,磷肥和钾肥以及有机肥全部基施),能增加马铃薯产量并能增加淀粉含量,从而提高马铃薯产品品质。在宁南山区马铃薯生产中可以推广应用这种肥料组配方案。

陕北黄土丘陵沟壑区水土保持耕作及施肥下农田土壤种子库特征

为了探明多年免耕下农田恶性杂草发生的机理,提高保护性耕作下作物对农田恶性杂草持久稳定的抑制效果,依据陕西安塞田间4a的定位试验,采用小区调查取样和室内实验相结合的方法,从物种组成、密度特征、多样性以及相似性特征等方面,研究了黄土丘陵旱作农区大豆(Glycine max)、玉米(Zea mays)、红小豆(Semen Phaseoli)、马铃薯(Solanum tuberosum)在翻耕化肥(CF)、翻耕有机肥(CM)、翻耕无肥(CN)、免耕化肥(NF)、免耕有机肥(NM)、免耕无肥(NN)等水平下的农田土壤种子库。结果表明:(1)4种作物24种土样中共萌发出12个物种1965株幼苗,隶属于7科12属。1年生杂草占94%,棒头草(fugax nees ex steud)、苋菜(Acalypha australis)、马唐(Digitaria sanguinalis)、早熟禾(Poasphondylodes)为优势种,占87%。(2)在0～20cm土层不同处理间,土壤种子库的密度变动于(282.9±63.4)～(7482.5±1078.3)粒.m-2,其中,红小豆小区>马铃薯小区>大豆小区>玉米小区;翻耕小区>免...

保水剂对宁南山区马铃薯产量及土壤水分利用的影响

在宁南半干旱黄土丘陵区探讨大田施用PAM、沃特保水剂对马铃薯产量和土壤水分利用的影响。结果表明:PAM、沃特均可促进马铃薯生长、提高块茎产量,其中PAM 9.0 kg/hm2(P3)和沃特15 kg/hm2(W1)、沃特30kg/hm2(W2)、沃特60 kg/hm2(W3)处理马铃薯生物量和块茎产量显著高于CK(P<0.05),增产效果最明显。幼苗期、块茎形成期和块茎生长期各处理耗水量无显著差异,淀粉积累期CK耗水量极显著高于施用保水剂处理(P>0.01)。保水剂处理耗水量随着马铃薯生物量的增加而增大,降雨对土壤水分的补充随生物量的增大而减少。利用有序聚类分析得出,淀粉积累期降雨对土壤0~60 cm的水分储量有明显的补充,施用PAM和沃特两种保水剂,0~140 cm各层土壤储水量恢复相近,而140 cm以下土层沃特的恢复能力优于PAM,平均每20 cm土层储水量较PAM高6.2 mm。随着PAM施用量的增加水分利用效率提高,沃特保水剂则相反。沃特15 kg/hm2和PAM 9 kg/hm2处理水分利用效率、水分产出效率最高。通过对马铃薯产量和水分利用特征得出,PAM的用量为9 kg/hm2、沃特15kg/h...

不同保水剂对宁南山区马铃薯生长发育和产量的影响

为促进宁南半干旱山区产业支柱作物马铃薯的生产,解决马铃薯生产、特别是苗期的干旱缺水问题,采用田间试验方法,进行PAM保水剂、多功能保水剂与一定配方尿素+过磷酸钙的施用对马铃薯生长发育、产量及效益的试验。结果表明,两种保水剂均能促进马铃薯生长发育,增加干物质积累,但是在不同生育时期促进作物生长的效果有所不同。1%PAM保水剂浸种2~3 min处理在前期效果显著,多功能保水剂在后期效果突出。马铃薯以施用多功能保水剂30 kg/hm2效果最好,其产量和商品薯分别比对照高出52.33%、138.29%,增收5 356.8元/hm2。研究表明,施用30 kg/hm2多功能保水剂比用1%PAM保水剂浸种2~3min更适宜宁南半干旱区及其同类地区旱地种植马铃薯。

鲜甘薯快速乙醇发酵条件的初步研究

本文对不同菌种（酵母菌和运动发酵单胞菌）快速生产燃料乙醇的条件进行了研究，实现了鲜甘薯快速转化为燃料乙醇。全文分为两部分： 第一部分：酵母菌快速生产燃料乙醇的条件研究。通过单因素试验，酵母菌快速生产燃料乙醇的条件为：发酵方式采用边糖化边发酵（SSF），蒸煮温度为85 ℃，料水比2:1（初始糖浓度 210 g/kg），糖化酶用量0.75 AGU/g 鲜甘薯，接种量10%（v/w）。在最优条件下，经过24 h发酵，乙醇浓度可达97.44 g/kg, 发酵效率为92%，发酵强度为4.06 g/kg/h。由于采用了低温蒸煮和SSF，可以大大节约能耗，从而降低乙醇生产的成本。同时，利用摇瓶优化的条件，进行了10 L，100 L，500 L发酵罐的放大试验，由于发酵罐初期可以人为通氧，使菌体能迅速积累，发酵时间缩短2 h，发酵效率在90%以上。 第二部分：运动发酵单胞菌快速生产燃料乙醇条件研究。通过单因素试验和正交试验获得了发酵的最佳参数：初始pH值6.0-7.0，硫酸铵5.0 g/kg，糖化酶量1.6 AUG/kg淀粉，初始糖浓度200 g/kg，接种量12.5%（v/w）。经过21 h发酵，乙醇浓度为95.15 g/kg，发酵效率可达94%。同时对不灭菌发酵也进行了研究，发酵效率可达92%。为鲜甘薯运动发酵单胞菌燃料乙醇的工业化生产打下基础。 对发酵结束后的残糖进行了研究。通过薄层层析和葡萄氧化酶测定证明：无论是酵母菌还是运动发酵单胞菌发酵结束后的发酵液中都不含葡萄糖。经过HPLC进一步分析残糖说明：发酵液中已没有葡萄糖成分；经糖化酶水解后仍没有葡萄糖出现；但经酸水解后又出现了葡萄糖，说明结束后的残糖是一些低聚糖结构。有关残糖的结构需要进一步研究。可以通过开发高效的低聚糖水解酶来降低发酵液的残糖，提高原料的利用率。 A new technology for rapid production fuel ethanol from fresh sweet potato by different microorganisms (Saccharomyces cerevisiae and Zymomonas mobilis) was gained in this research. The paper involved two parts: Part 1: The study on fuel ethanol rapid production from fresh sweet potato by Saccharomyces cerevisiae. The following parameters of Saccharomyces cerevisiae was investigated by a series of experiments: fermentation models, cooking temperature, initial sugar concentration and glucoamylase dosage. The results showed that SSF (simultaneous saccharification and fermentation) not only reduced the fermentation time (from 30 to 24h) but also enhanced the ethanol concentration (from 73.56 to 95.96 g/kg). With low-temperature-cooking (85 ℃) using SSF, the Saccharomyces cerevisiae was able to produce ethanol 97.44 g/kg which the fermentation yield could reach to 92% and ethanol productivity 4.06 g/kg/h from sweet potato enzymatic hydrolysis. Furthermore, the savings in energy by carrying out the cooking (85 ℃) and saccharification (30 ℃) step at low temperature had been realized. The results were also verified in 10 L, 100 L and 500 L fermentor. The fermentation yield was no less than 90%. The fermentation time of fermenter was shorter than Erlenmeyer flask. This may be that the aeration in the early fermentation period is available, which lead to the rapidly commutations of biomass. Part 2: The technology of ethanol rapid production with simultaneous saccharification and fermentation ( SSF ) by Zymomonas mobilis，using fresh sweet potato as raw material was studied. The effects of various factors on the yield of ethanol were investigated by the single factor and the orthogonal experiments. As a result, the optimal technical conditions were obtained from those experiments：initial pH value 6.0-7.0, nitride 5.0 g/kg，(NH4)2SO4, glucoamylase 1.6 AUG/kg starch, inoculums concentration 12.5% (v/w). The Zymomonas mobilis was able to produce ethanol 95.15 g/kg, with 94% of the theoretical yield, from fresh sweet potato after 24 h fermentation. The fermentation efficiency of non-sterilized was also reach to 92%. We also analyzed the final fermentation residual sugars of Saccharomyces cerevisiae and Zymomonas mobilis. When the residual sugars were analyzed by thin-layer chromatogram and glucose oxidase, there was no glucose. The analysis of reducing sugars by HPLC showed that there was no glucose existed in the fermentation liquor. However, the glucose appeared after being hydrolyzed by acid. It is indicated that the residual sugars in the final fermentation liquor were the configuration of oligosaccharide, which was linked by the special glycosidic bonds. It was feasible for reducing residual sugars to develope the enzyme that can degradation the oligosaccharide.

毛壳菌产抗真菌活性物质菌株筛选及种间原生质体融合研究

毛壳菌属很多种类具有重要生防价值，其生防机理包括对植物病原真菌的重寄生作用、诱导植物产生抗病性、产生抗真菌活性的次生代谢产物等。迄今，学界对毛壳菌的研究主要集中在毛壳菌的生防机理，毛壳菌活性次生代谢产物的分离等方面。本研究致力于产抗生素的毛壳菌的种间原生质体融合，从产抗生素毛壳菌菌株的筛选开始，进而对产抗生素的角毛壳菌进行诱变选育，最终用产不同抗生素的角毛壳菌与球毛壳菌进行种间原生质体融合。主要有以下五方面研究结果。 1、毛壳菌抗真菌活性物质产生菌株的筛选：不同毛壳菌菌株发酵液采用琼脂扩散法对植物病原真菌进行抑菌活性试验，结果显示，菌株CH08和CH23的发酵液对芒果炭疽、苹果炭疽和马铃薯晚疫菌具有抑制作用。菌株CH16和CH17的发酵液对芒果炭疽菌、苹果炭疽菌有抑制作用。菌株CH21发酵液对辣椒炭疽菌和西瓜枯萎菌有抑制作用。经形态学研究，菌株CH08、CH16、CH17和CH23鉴定为球毛壳菌，菌株CH21鉴定为角毛壳菌。对角毛壳菌与球毛壳菌菌株发酵液抑菌谱比较，发现角毛壳菌与球毛壳菌发酵液具有明显不同的抑菌谱，表明角毛壳菌与球毛壳菌产生不同的抗真菌活性物质。 2、角毛壳菌（CH21）和球毛壳菌（CH08）原生质体制备和再生条件研究：考察了菌龄、酶浓度、稳渗剂及其浓度、酶解温度、酶解时间及再生培养基对原生质体制备和再生的影响。用菌龄为生长54 h的角毛壳菌菌丝，以0.06 M磷酸缓冲液（pH6.0）配制成含蜗牛酶15 mg/ml、溶壁酶10 mg/ml、蔗糖0.6 mol/L的酶解液，30℃酶解1.5 h，原生质体释放量2.02×107个/g；以PDA为再生培养基，0.7 mol/L的蔗糖再生稳渗剂，再生率可达51.45%。用菌龄为生长48 h的球毛壳菌菌丝，以0.06 M磷酸缓冲液（pH6.0）配制成含蜗牛酶15 mg/ml、溶壁酶10 mg/ml、蔗糖0.6 mol/L的酶解液，30℃酶解1 h，原生质体释放量达1.57×108个/g；以PDA为再生培养基，0.7 mol/L的蔗糖为再生稳渗剂，再生率可达41.48%。 3、角毛壳菌（CH21）原生质体紫外诱变选育：以CH21为出发菌株，制备原生质体进行紫外诱变，诱变条件为：15 w紫外灯，距离30 cm，照射90 s，致死率80%~85%。建立了诱变菌株初筛的双层平板筛选模型。经平板初筛和摇瓶复筛，获得一株突变菌株CH21-I-402，其发酵液抑菌活性较出发菌株提高18.3%。 4、抗性标记菌株的获得：菌株CH21-I-402和CH08抗生素药敏试验表明， CH21-I-402菌株对潮霉素有抗性、对G418（Geneticin）敏感，菌株CH08对潮霉素和G418都敏感。根癌农杆菌EHA105介导的新霉素磷酸转移酶基因转化球毛壳菌，经PCR检测，新霉素磷酸转移酶基因成功转化进菌株CH08-GR70，CH08-GR120。转化子对G418抗性提高3～4倍，对潮霉素仍然比较敏感。 5、以G418和潮霉素抗性为筛选标记的原生质体融合与融合菌株AFLP分析：制备角毛壳菌CH21-I-402和球毛壳菌CH08-GR70原生质体，以35％的PEG6000为助融剂进行原生质体融合，以65 μg/ml的潮霉素和60 μg/ml G418为抗性筛选标记，获得46个再生菌株。再生菌株连续传代5代后，再生菌株表现出多种形态类型。利用AFLP技术对再生菌株及亲本菌株基因组DNA分析表明，再生菌株PF1、PF26为融合菌株。抑菌活性测试表明，融合菌株PF26发酵液对芒果炭疽菌和苹果轮纹菌有强的抑制作用，且抑菌活性比亲本球毛壳菌明显提高。 Chaetomium spp. have great potentials as biocontrol agents against a range of plant pathogens on the basis of its mycoparasitism, induced plant disease resistance, production of antifungal metabolites, and so on. Previous researches on C. spp. mostly focused on the mechanisms of its biocontrol and the isolation of secondary metabolites. In this study, screening antifungal C. spp., mutation breeding of C. cupreum and interspecies protoplast fusion between C. cupreum and C. globosum were carried out, respectively. The corresponding results are as follows: Firstly, among more than 40 C. spp., the strains produced anti-fungal antibiotics were screened by agar diffusion experiments. Results showed that both CH08 and CH23 had inhibition against Colletotrichum gloeosporioides, Cladosporium fulvum, and Phytophthora infestans. Both CH16 and CH17 had inhibition against Colletotrichum gloeosporioides and Cladosporium fulvum. In addition, CH21 exhibited anti-fungal activity against Fusarium oxysporum f. sp niveum and Colletotrichum capsici. Furthermore, CH08, CH16, CH17 and CH23 were identified as C. globosum, CH21 was proved to be C. cupreum based on morphology. The comparison of the anti-fungal spectrum between C. cupreum and C. globosum, showed they could produce different antibiotics. Secondly, specified protocols for preparing and regenerating protoplasts from mycelia of C. cupreum CH21 and C. globosum CH08 were studied. The effects of the age mycelia, the concentration of enzyme, digestion temperature and time, kinds of osmotic stabilizer and regeneration medium on protoplasts preparation and regeneration were all optimized, respectively. In one protocol, with 15 mg/mL snailase, 10 mg/mL lywallzyme, 0.6 M sucrose, in 0.06 M phosphate buffer (pH6.0), and digested for 1.5 h at 30 ºC, 2.02×107 protoplasts from each gram mycelia were obtained from cultures of C. cupreum CH21 grown in potato dextrose broth (PDB) medium for 54 h. And when 0.7 M sucrose was used as osmotic stabilizer in the regeneration medium OPDA (potato dextrose agar with osmotic stabilize), the regeneration efficiency of protoplasts was 51.45%. In another protocol, with 15 mg/mL snailase, 10 mg/mL lywallzyme, 0.6 M sucrose, in 0.06 M phosphate buffer (pH6.0), and digested for 1 h at 30 ºC, 1.57×108 protoplasts from each gram mycelia were obtained from cultures of C. globosum CH08 grown in PDB for 48 h. And when 0.7 M sucrose was used as osmotic stabilizer in the regeneration medium OPDA, the regeneration efficiency of protoplasts was 41.48%. Thirdly, the mutagenesis conditions and secondary screening model of C. cupreum CH21 were explored. An 80% to 85% death rate could be achieved when the protoplasts of C. cupreum CH21 were irradiated by 15 w UV lamp from 30 cm distance for 90 s. In addition, the doublelayer plate’s method for the primary screening of high-producing antibiotics strains was established. A high yielding antibiotic mutant CH21-I-402 was obtained through the primary screening on plate and the secondary selection in Erlenmeyer flask, compared to the original CH21 strain, the antifungal activity of the mutant CH21-I-402 was increased by 18.3%. Fourth, the sensitivity to antibiotics of both C. cupreum CH21-I-402 and C. globusm CH08 was detected. Results showed C. cupreum CH21-I-402 was sensitive to G418 (Geneticin) (Gs) and resistant to Hygromycin B(Hr), and C. globusm CH08 was sensitive to both G418 (Geneticin) (Gs) and Hygromycin B(Hs). At the same time, neomycin phosphotransferase II (npt II) gene was transformed into C. globusm CH08(Gs, Hs) mediated by Agrobacterium tumefaciens EHA105, and the npt II gene was verified by polymerase chain reaction in resistance to G418 strains CH08-GR70 and CH08-GR120. The transformants still showed sensitive to Hygromycin B(Hs). Finally, a selection system for hybrids was set up by interspecies protoplast fusion between C. cupreum and C. globusm using dominant selective drug resistance markers. At first, protoplasts of C. cupreum CH21-I-402 (Hr, Gs) and C. globusm CH08-GR70 (Hs, Gr) were prepared, then the protoplasts were fused in the presence of 35% polyethylene glycol 6000 and regenerated on OPDA medium with 65 μg/ml Hygromycin B and 60μg/ml G418, at last 46 colonies with Hr and Gr were obtained. Even after 5 generations’ subculture, most of the colonies displayed significant difference in taxonomic characteristics with their parental strains. Regenerated strains PF1 and PF26 were confirmed as fusants by amplified fragment length polymorphisms analysis with the genomic DNA as the model. PF26 showed higher inhibitory activity against Colletotrichum gloeosporioides and Macrophoma kuwatsukai than that of the parental strain C. globusm.

一株掷孢酵母利用红薯淀粉酶解液积累油脂研究

本论文以红薯淀粉的双酶法水解液为碳源，从19 株红色酵母中筛选出一株油脂含量较高的菌株掷孢酵母（Sporobolomyces reseus）As.2.618。为了提高掷孢酵母（S.reseus）As.2.618 的油脂产量，考察了培养基组成对该菌生长情况及油脂积累的影响。用均匀设计法对培养基组成进行了优化，由DPS软件得出的优化结果为：还原糖103g/L、酵母粉11.5g/L、磷酸二氢钾0.3g/L、硫酸镁0.15g/L。生物量可达19.23 g/L,油脂含量为3.875 g/L。研究了添加二价离子对该菌的生长及油脂积累的影响,结果表明Zn2+对该菌生长和油脂积累都有显著促进作用。研究了发酵条件以及添加氧载体正十二烷对该菌发酵的影响，表明添加正十二烷有利用于该菌生长与油脂积累。得出最佳发酵条件是：在还原糖103g/L、酵母粉11.5g/L、磷酸二氢钾0.3g/L、硫酸镁0.15g/L。添加30mg/L 硫酸锌,接种量为5％，在24h 后添加2g/L 的碳酸钙和2%(v/v)正十二烷，pH6.0 培养温度为27℃，转速为200r/min，培养时间为7 天的条件下，该菌生物量干重可达35.05g/L,油脂含量也达11.98g/L。Lipid is one of the basic material for life-sustaining activities andimportant industrial materials. As lipid resources mainly come from the animal andthe plant, the problem of lipid lack is encountered at times. The lipid frommicroorganisms is the substitute and superior to the above lipid with a short period ofproduction and much cheaper fermentation materials such as agricultural and sidelineproducts or wastes of crop.Thus large scale production and broad application ofmicrobial lipid will be efficient not only in substitute of the animal and the plant lipidfor food and industrial field , but also inducing a new way leading to solve the energyproblem.For the purpose of exploring the characteristics of lipid production of redyeasts from sweet potato starch hydrolysates. 19 red yeasts are screened for thecapability of lipid producing and one strain Sporobolomyces reseus As.2.618 withsuperior performance is sellected.To improve the Sporobolomyces reseus As.2.618’s capability of lipidaccumulation , the components of the medium, which may influence the growth of thestrain and the lipid yield have been studied. To get the optimum mediumcomponents ,the “uniform design” was used .The DPS software gave the optimummedium component is: reducing sugar 103 g/L、yeast extract 11.5 g/L、KH2PO4 0.3g/L、MgSO4 0.15 g/L. The biomass could reach up to 19.23 g/L and lipid yield 3.87g/L with the above composition of fermentation medium.Furthermore the fermentation conditions , addition of the divalent metal ionsand the oxygen vector to increase the strain’s lipid producing capability are tested.The optimum condition is : reducing sugar 103 g/L、yeast extract 11.5 g/L、KH2PO40.3 g/L、MgSO4 0.15 g/L，Adding 30mg/L ZnSO4,and adding 2g/L CaCO3 2％(v/v)n-dodecane after 24h’s fermentation. the optimal fermentation condition were asfollow :30ml medium in the 500ml flask with initial pH 6.0,the flasks with 5%inoculation volume were at 200r/min shaking speed for 7d’s fermentation at27 .Under this kind of condition the high biom ¡æ ass which reach to 35.05 g/L could begot ,the yield of lipid also could reach to 11.98g/L.

微生物酶法拆分消旋芳香族氨基酸的研究

通过单因子和多因子摇瓶正交试验，确定了米曲霉液态发酵产氨基酰化酶的最佳发酵条件。优化发酵培养基组成（ρ/g L-1）： 葡萄糖40,蔗糖10,可溶性淀粉20,蛋白胨2.5,马铃薯液1 000mL, pH自然。培养基装量50mL/250mL三角瓶，接种量4%。培养温度30℃，转速100 rmin-1，发酵时间42h。每50mL培养物的总酶活由优化前的2627U提高到7338U，是优化前的2.79倍。 研究了米曲霉氨基酰化酶的部分酶学性质，该酶催化反应的最适pH为7.0，最适温度为40℃，低浓度的Co2+（5×10-4mol/L）对酶活激活作用显著，催化反应过程中，底物浓度大于0.2 mol/L时，存在高浓度底物抑制酶活力现象。 初步探索了包埋法固定化米曲霉氨基酰化酶的载体，在实验的五种载体中，以海藻酸钠为载体包埋固定化米曲霉氨基酰化酶酶活保留率高，且操作简单，成本低廉。对包埋法固定化米曲霉氨基酰化酶酶学性质进行了研究，较游离米曲霉氨基酰化酶，最适温度未发生改变，最适pH向碱性范围偏移至8.0，对酸碱和热的稳定性增强，最适底物浓度增大到0.4 mol/L。 根据氨基酰化酶能立体专一水解L-氨基酰化物的特点，利用米曲霉氨基酰化酶对消旋苯丙氨酸进行了拆分。在米曲霉氨基酰化酶选择性的作用于底物N-乙酰-L-苯丙氨酸，得到L-苯丙氨酸后，通过732阳离子树脂和结晶法分别将L-苯丙氨酸和N-乙酰-D-苯丙氨酸分离，N-乙酰-D-苯丙氨酸通过酸水解脱去乙酰基得到D-苯丙氨酸，拆分得到光学纯度为98%的L-苯丙氨酸（收率84.8%）和光学纯度为92.3%的D-苯丙氨酸（收率89.5%）。 separate factors tests and orthogonal experiments,the optimum fermentation conditions of aminoacylase –producing Aspergillus oryzae were determined, as follows（ρ/g L-1）,glucose 40,sucrose 10,soluble starch 20,peptone 2.5，potato juice 1000ml, inoculation volume 4%and fermentation temperature 30℃,rotation speed 100rmin-1.The highest total enzyme activity ,7338μ,was obtained after fermentation for 42 h, increased by 279% compared with the original value of 2627μbefore optimization. We dicussed partial characteristics of aminoacylase. The optimal pH and temperature of aminoacylase were 7.0 and 40℃ respectively. Low- concentration Co2+ (5×10-4mol/L）activated the aminoacylase remarkably while high-concentration substrate lowered the aminoacylase . Five vectors has been used for immobolizing the enzyme and calcium alginate showed to be the best one for it had the slightest influence on the enzyme activity, easy to operate ,and low in price, comparing with other fours. The enzymatic charateristic study showed that its optimum temperature didn’t change, but the optimum pH and substrat concentration were higher after immobilization. The stability of immobolized enzyme to acid, alkaline and heat rised as well. The aminoacylse from Aspergillus oryzae was used to resolute racemic phenylalanine to obtain D-phenylalanine. After catalyzing process, we took two methods to separate D-phenylalanine .In end,L-phenylalanine was obtained with 98% optical purity in 84.8% yield, D-phenylalanine was obtained with 92.3% optical purity in 89.5% yield.

非粮原料燃料乙醇发酵关键技术的研究

生物质燃料乙醇是一种高度清洁的交通液体燃料，是减少温室气体排放，缓解大气污染的最佳技术选择。以非粮原料生产燃料乙醇可以在进行能源生产的同时保证粮食安全，有利于产业的可持续发展。在众多的非粮原料中，甘薯是我国开发潜力最大的生物质能源作物之一。我国占世界甘薯种植总面积和产量的90％。同时，甘薯的单位面积燃料乙醇产量远大于玉米和小麦。其成本是目前酒精中最低廉的，因此利用甘薯生产乙醇是发展生物质燃料乙醇的首要选择。目前采用薯类全原料主要采用分批发酵生产乙醇，其技术水平低，发酵强度低，一般在0.7－2.5g/（L•h），乙醇浓度低，甘薯发酵乙醇为6－8％(v/v)，能耗高，环境负荷大，污染严重。针对上述问题，本文从菌株选育、原料预处理、中试放大、残糖成分分析等方面进行研究。 为了研究乙醇发酵生产规模扩大过程中，大型发酵罐底部高压条件下，CO2对酵母乙醇发酵的影响，我们通过CO2 加压的方法进行模拟试验，研究结果表明，发酵时间随压强的升高而逐渐延长，高压CO2 对乙醇发酵效率影响不大，在0.3 MPa 以下时，发酵效率均可达到90%以上。高压CO2 对发酵的抑制作用是高压和CO2 这两个因素联合作用的结果。高压CO2 条件下，酵母胞外酶和胞内重要酶类的酶活均表现出特征性。0.2 MPa 下，酶活性的变化趋势和0.1 MPa 条件下的较为一致。而0.3 MPa 下的酶活变化趋势与0.4 MPa 下的酶活更为接近。通过全基因表达分析发现在CO2 压力为0.3 MPa 下，乙醇发酵途径中多个基因表达量下调，同时海藻糖合成酶和热激蛋白基因表达量上调。 筛选耐高温的乙醇酵母菌株能够解决糖化温度和发酵温度不协调的矛盾，实现真正意义上的边糖化边发酵。高温发酵还能够降低发酵时的冷却成本，实现乙醇的周年生产。本研究筛选出一株高温发酵菌株Y-H1，进而我们对该菌株的胞外酶和胞内乙醇代谢重要酶类的酶活性进行了分析。结果表明Y-H1 能够在40 ℃条件下正常进行乙醇发酵，发酵33h，最终乙醇浓度达到10.7%（w/w），发酵效率达到90%以上。同时发酵液最终pH 在3.5 左右，显示菌株具有一定的耐酸性能力。同时观察到40 ℃下，菌株的胞外酶和胞内乙醇代谢重要酶类的酶活性发生了变化，乙醇发酵途径中关键酶基因表达下调，而海藻糖合成酶与热激蛋白基因表达量上调，这些结果为进一步研究酵母菌耐热调控机理提供了依据。 糖蜜是一种大规模工业生产乙醇的理想原料，本研究利用选育高浓度乙醇发酵菌株结合配套的发酵稳定剂，研究了糖蜜高浓度乙醇发酵情况。结果表明采用冷酸沉淀预处理糖蜜溶液，采用分批补料的发酵方式，乙醇浓度最高达到了10.26% (w/w)，发酵时间为42 h。同时观察到在糖蜜发酵中，乙醛含量与乙醇浓度存在一定的相关性。 快速乙醇发酵对于缩短乙醇生产周期、降低乙醇生产成本、减少原料腐烂损失具有重要意义。本研究诱变和筛选得到了一株快速乙醇发酵菌株10232B。在优化后的发酵条件下，采用10L 发酵罐进行分批乙醇发酵，经过18h,乙醇的最终浓度达到88.5g/L,发酵效率93.6%，平均乙醇生产速度达到4.92 g/L/h。此菌株在保持较高乙醇生产浓度的同时，拥有快速生产乙醇的能力，适合作为快速乙醇发酵生产菌种。 由于鲜甘薯具有粘度大的特点，传统液化糖化处理很难在短时间内充分糖化原料；高粘度的醪液也难以进行管道输送，容易堵塞管路；同时，也会降低后续的乙醇发酵效率。 本文采用了快速粘度分析法对鲜甘薯糊化粘度特性进行了分析，进而对预处理条件进行了研究，在最佳预处理条件下，糖化2h 后，醪液葡萄糖值最高可达99.3，粘度4.5×104 mPa.s，而采用传统糖化工艺，醪液DE 值仅为85.8，粘度大于1.0×105 mPa.s。 此预处理方法也可用于快速糖化不加水的醪液。后续的乙醇发酵试验表明，通过此预处理方法获得的糖化醪液对乙醇发酵无负面影响。 在前期已实现了实验室水平的鲜甘薯燃料乙醇快速乙醇发酵基础上，进一步将发酵规模扩大到500L，在中试水平上对甘薯乙醇发酵进行了研究。结果表明在500L 中试规模，采用边糖化边发酵（SSF）工艺，在料液比为3∶1，发酵醪液最高粘度为6×104mPa.s 条件下，发酵37h，乙醇浓度达到了12.7%（v/v），发酵效率91%，发酵强度为2.7 g/（L•h）。与目前国内的薯类乙醇发酵生产技术水平具有明显的优越性。 为研究甘薯、木薯乙醇发酵中残糖的组成，采用了高效液相色谱—蒸发光散射检测法，对乙醇发酵残糖进行了分析。结果表明，甘薯、木薯乙醇发酵残糖均为寡聚糖，主要由葡萄糖、木糖、半乳糖、阿拉伯糖和甘露糖构成。随着发酵时间延长，寡聚糖中的葡萄糖、半乳糖、甘露糖可被缓慢的水解释放。提高糖化酶量仅在一定程度上降低残糖，过量的糖化酶反而会导致残糖增加。同时发现3, 5-二硝基水杨酸法不能准确测定甘薯、木薯乙醇发酵中的残总糖含量。进一步筛选了两株残糖降解菌株，对甘薯乙醇发酵残糖的降解利用率均达到了40%以上，而且还能显著降低发酵醪液粘度。经形态学和rRNA ITS 序列分析，确定这两株菌分别属于为木霉属和曲霉属黑曲霉组。 通过对以甘薯原料为代表的非粮原料发酵技术研究开发，以期形成乙醇转化率高，能耗低，生产效率高、季节适应性好，原料适应性广，经济性强，符合清洁生产机制的燃料乙醇高效转化技术，为具有我国特色的燃料乙醇发展模式提供技术支持。 Sweet potato is one of the major feedstock for the fuel ethanol production in China. The planting area and the yield in China take 90% of the world. Sweet potato is an efficient kind of energy crops. The energy outcome per area is higher than corn or wheat. And the manufacture cost of ethanol is the lowest, compared with corn and wheat. So sweet potato is the favorable crop for the bioethanol production in China. However, the low-level fermentation technology restricts the development of ethanol production by sweet potato, including slow ethanol production rate, low ethanol concentration and high energy cost. To solve these problems, we conducted research on the strain breeding, pretreatment, pilot fermentation test and residual saccharides analysis. To study the impact of hyperbaric condition at bottom of the large fermentor on yeast fermentation, high pressure carbon dioxide (CO2) was adopted to simulate the situation. The results showed that the fermentation was prolonged with the increasing pressure. The pressure of CO2 had little impact on the ethanol yield which could reach 90% under the pressure below 0.3 MPa. The inhibition was combined by the high pressure and CO2. Under the high CO2 pressure, the extracellular and important intracellular enzyme activities were different from those under normal state. The changes under 0.1 MPa and 0.2 MPa were similar. The changes under 0.3 MPa were closer to those under 0.4 MPa. The application of thermotolerance yeast could solve the problem of the inconsistent temperature between fermentation and saccharificaton and fulfill the real simultaneous saccharification and fermentation. And it could reduce the cooling cost. A thermotolerance strain Y-H1 was isolated in our research. It gave high ethanol concentration of 10.7%（w/w）at 40 ℃ for 33 h. The ethanol yield efficiency was over 90%. At 40 ℃, the extracellular and important intracellular enzyme activities of Y-H1 showed the difference with normal state, which may indicate its physiological changes at the high temperature. Molasses is another feedstock for industrial ethanol production. By our ethanol-tolerance strain and the regulation reagents, the fermentation with high ethanol concentration was investigated. In fed-batch mode combined with cold acid deposition, the highest ethanol concentration was 10.26% (w/w) for 42h. The aldehyde concentration in fermentation was found to be related to ethanol concentration. The development of a rapid ethanol fermentation strain of Zymomonas mobilis is essential for reducing the cost of ethanol production and for the timely utilization of fresh material that is easily decayed in the Chinese bioethanol industry. A mutant Z. mobilis strain, 10232B, was generated by UV mutagenesis. Under these optimized conditions, fermentation of the mutant Z. mobilis 10232B strain was completed in just 18 h with a high ethanol production rate, at an average of 4.92 gL-1h-1 per batch. The final maximum ethanol concentration was 88.5 gL-1, with an ethanol yield efficiency of 93.6%. This result illustrated the potential use of the mutant Z. mobilis 10232B strain in rapid ethanol fermentation in order to help reduce the cost of industrial ethanol production. As fresh sweet potato syrup shows high viscosity, it is hard to be fully converted to glucose by enzymes in the traditional saccharification process. The high-viscosity syrup is difficult to be transmitted in pipes, which may be easily blocked. Meanwhile it could also reduce the later ethanol fermentation efficiency. To solve these problems, effects of the pretreatment conditions were investigated. The highest dextrose equivalent value of 99.3 and the lowest viscosity of 4.5×104 mPa.s were obtained by the most favorable pretreatment conditions, while those of 85.8 and over 1.0×105 mPa.s was produced by traditional treatment conditions. The pretreatment could also be applied on the material syrup without adding water. The later experiments showed that the pretreated syrup had no negative effect on the ethanol fermentation and exhibited lower viscosity. The fuel ethanol rapid production from fresh sweet potato was enlarged in the 500L pilot scale after its fulfillment on the laboratory level. The optimal ratio of material to water was 3 to 1 in 500L fermentor. With low-temperature-cooking (85 ℃) using SSF, the Saccharomyces cerevisiae was able to produce ethanol 97.44 g/kg for 37h, which reached 92% of theoretical yield. The average ethanol production rate was 4.06 g/kg/h. And the maximum viscosity of syrup reached 6×104mPa.s. The results showed its superiority over current industrial ethanol fermentation. The compositions of the residual saccharides in the ethanol fermentation by sweet potato and cassava were analyzed by high performance liquid chromatography coupled with evaporative light-scattering detector. The results showed that all the residual saccharides were oligosaccharides, mainly composed of glucose, xylose, galactose, arabinose and mannose. The glucose, galactose and mannose could be slowly hydrolyzed from oligosaccharides in syrup during a long period. To increase the glucoamylase dosage could lower the residual saccharides to a certain extent. However, excess glucoamylase dosage led to more residual saccharides. And the method of 3, 5-dinitrosalicylic acid could not accurately quantify the residual total saccharides content. Two residual saccharides degrading strains were isolated, which could utilize 40% of total residual saccharide and lower the syrup viscosity. With the analysis of morphology and internal transcribed spacer sequence, they were finally identified as species of Trichoderma and Aspergillus niger.