小鲵属分子系统发育研究

小鲵属为亚洲特有的有尾两栖类，是小鲵科之模式属。现记载小鲵属动物有29种，占全科物种数一半以上（Frost, 2007），为小鲵科第一大属。该属分布跨越古北界和东洋界，分布于中国、朝鲜、韩国、日本等地区，其系统学研究一直以来颇为中外学者所关注。澄清该属的物种分类问题，阐明其种间的系统发育关系对整个小鲵科的系统演化与分布格局关系的研究具有关键性意义。 本论文以中国及周边地区的小鲵属物种为主要对象，主要利用分子生物学实验与生物信息学途径相结合的手段，运用支序系统学与分子进化生物学理论及分析方法，展开系统发育的研究。在此基础上诠释现存的分类问题，并探讨该属系统发育关系。 研究材料上，本研究采用野外采集与网络下载数据相结合的方法，获取了较为全面的小鲵属物种DNA序列资料。技术手段上，选取了线粒体DNA的Cytb、12S、16S、NADH 2、COI等多个基因部分片段序列，对小鲵属开展了较为全面系统的研究。分析方法上，针对小鲵属物种各类群的具体情况，运用了处于领域前沿的多种分析方法。应用PAUP、MrBayes、Modeltest、Mega等软件，采用了最大简约法（MP）、邻接法（NJ）、贝叶斯推断（BI）及K2P遗传距离分析等方法。 本研究对小鲵属进行了较为全面的系统发育研究，弥补了有关小鲵属系统发育研究的不足，并得出了以下结果： （1）关于豫南小鲵Hynobius yunanicus的有效性，基于细胞色素b序列的系统发育关系联合形态和染色体组型等证据证明了豫南小鲵是商城肥鲵的同物异名。 （2）获得了较为全面的小鲵属物种系统发育树，并以此解释了北海道滞育小鲵、东北小鲵、中国小鲵与义乌小鲵等存在的分类问题。 （3）本研究利用DNA条形码技术对小鲵属及小鲵科物种进行了鉴定，再次证明豫南小鲵为商城肥鲵的同物异名；并认为猫儿山小鲵与挂榜山小鲵为同物异名。 综上，本研究较为完整地勾勒了小鲵属的系统发育关系全貌，并对小鲵属物种的起源进行了推测。 Hynobius, the type genus of the Family Hynobiidae, is the only exclusively Asian salamander genus. This genus which contains 29 species (beyond half of total Family), is the key group in Hynobiidae. The genus distributed across Palaearctic and Oriental Realm, and was found in China, Korea, and Japan. Systematics of genus Hynobius draws attention of researchers all the times. Resolving the taxonomic and phynogenetic questions of Hynobius is very important to the evolutionary research of Family Hynobiidae. Firstly, studies on systematics of genus Hynobius based on morphology, karyotype and molecular phylogeny of Hynobius are reviewed along with existing questions of this genus. The sequential reaserch project of phylogenetics is perspectively outlined. Using molecular data, we compared Hynobius yunanicus with a sympatric species Pachyhynobius shangchengensis. Our cytb sequences associating with karyotypic and morphological data supportted that H. yunanicus is not a valid species, but a synonym of P. shangchengensis. Because of phenotypic plasticity, some morphological characters are not even suitable for identifying hynobiids. The taxonomy of hynobiids is still controversial to a certain extent (Zhao et al. 1993; Fei, 1999; Chen et al. 2001; Zeng et al. 2006) and needs to be resolved by a new method. Here we examined the utility of COI barcoding for the discrimination of hynobiids. Meantime, the taxonomy of this Family was looked-over again. Our result show that the DNA Barcoding based on COI is easier and more rapidly than classic methods. And the DNA Barcodes data supported the actual taxonomy of Hynobiidae. Based on the achievements of our research, the phylogeny of Hynobius was reconstructed including some new species (H. maoershanensis, H. guabangshanensis, etc). Besides the phylogenetics of Hynobius was outlined, some questions and the hypothesis about the origin of genus Hynobius was put out.

两株耐碱性木聚糖酶产生菌的筛选及产酶条件研究

本文主要研究了从造纸厂碱性土壤中筛选得到的，能够产生耐碱木聚糖酶的两株放线菌X24-14和X15-17。通过16 S rRNA基因序列分析并结合菌株的形态特征以及生理生化特性，初步认为菌株X15-17为拟诺卡氏菌属（Nocardiopsis）的一个潜在新种；菌株X24-14为纤维化纤维菌（Cellulosimicrobium cellulans）。 在此基础上探索了菌株X24-14和菌株X15-17所产木聚糖酶的基本酶学性质。研究发现，两株菌所产的木聚糖酶的耐碱性均较强： 1）菌株X24-14所产的木聚糖酶，在pH 4.2～9.4的范围内能维持较高的活力，pH 9.4条件下，仍能保持80％的酶活力；2）菌株X15-17所产的木聚糖酶在pH 4.0～9.0的范围内能维持较高的活力，pH 9.0条件下，仍能保持80％的酶活力；3）两株菌所产的木聚糖酶均具有较好的pH稳定性，在pH 2.0～11.0范围内稳定，pH 11.0、4 ℃条件下处理24 h仍具有75％的活力。 本文还重点研究了菌株X24-14在不同培养基成分及不同培养条件下的产酶情况，确定了其适宜的产酶条件。结果显示，菌株X24-14的最适碳源为麸皮；最适氮源为蛋白胨；最适产酶pH为pH 8.5。菌株X24-14适宜的产酶条件为：麸皮60 g/L，蛋白胨10 g/L，K2HPO4 7.0 g/L，pH 8.5，接种量为5％，37 ℃，200 r/min发酵培养108 h。 Two strains of actinomycetes, X24-14 and X15-17, which produced alkali-tolerant xylanase were screened from the soil samples collected from a pulp mill in china. Based on the morphological, physiochemical characteristics and 16S rRNA sequence, X24-14 was priminarily identified as cellulosimicrobium cellulans ; X15-17 was priminarily identified as a new species of Nocardiopsis. The investigation examined the enzyme activities which produced by X24-14 and X15-17 under different pH and different temperatures. The results showed that : 1）The xylanase from X24-14 had characteristic of alkali-tolerance: It remains 80% relative activity at pH ranges between pH 4.2 and pH 9.4 under 50℃. 2)The xylanase from X15-17 also showed characteristic of alkali-tolerance, it remains 80% relative activity at pH ranges between pH 4.0and pH 9.0 under 50℃. 3）The xylanase from the two strains showed alkali-stable characteristics. They were stable at pH ranges between pH 2.0 and pH 11.0, showing 75% of its maximal activity remaining under 24 hours of treatment at 4℃. We also studied the effect of different growth conditions: carbon source, nitrogen sources, inoculum size, and initial pH on the production of xylanase of strain X24-14. The results showed that :The optimal carbon source was wheat bran; The optima nitrogen source was peptone; The maximum xylanase activity was achieved in the medium containing 60 g/L wheat bran, 10 g/L peptone, 7 g/L K2HPO4, inoculum size 5% and pH 8.5, under 37℃ in 108 h.

猪场废水短程硝化反硝化-厌氧氨氧化脱氮研究

畜禽废水是农村水环境污染的主要来源之一，其处理的难点在于脱氮。传统生物脱氮法具有能耗高、需大量外加碳源等缺点，开发低成本、高效率的新型生物脱氮技术具有重要意义。 本研究将短程硝化反硝化和厌氧氨氧化两种脱氮新技术结合，让前者为后者创造去除可降解COD、降低总氮负荷、调整pH、调整氨氮和亚硝酸盐氮浓度比例等进水条件，而后者可在无需外加碳源的条件下进一步脱氮，二者结合可成为高氨氮、低C/N废水脱氮的新途径。 试验以低碳氮比猪场废水为研究对象，首先进行了短程硝化反硝化预处理研究，同时启动并运行调控厌氧氨氧化反应器，最后以经过短程硝化反硝化预处理的猪场废水为进水，进行厌氧氨氧化脱氮考察。实验表明：（1）短程硝化反硝化作为厌氧氨氧化的预处理工序是可行的。猪场废水通过短程硝化反硝化，可以达到基本去除可生化COD、部分脱氮、控制出水氨氮和亚硝酸盐氮浓度之比在1︰1左右、pH在7.5～8.0的目的， COD和总氮平均去除率分别为64.3%、49.1%,出水可达到厌氧氨氧化反应的进水要求。（2）采用模拟废水启动厌氧氨氧化反应器，经过5个月左右的运行调控，反应器启动成功并稳定运行，最高总氮去除率为87.1%，总氮容积去除率最高达到0.14kg/m3.d；整个稳定阶段，氨氮、亚硝酸盐氮、硝酸盐氮的变化量之比为1︰1.21︰0.33。（3）经过短程硝化反硝化预处理的猪场废水厌氧氨氧化脱氮效果稳定，氨氮、亚硝酸盐氮、总氮、COD的平均去除率分别为93.0%、99.4%、84.6%、18.1%，处理效果与模拟废水处理系统相比无明显变化。（4）经过短程硝化反硝化预处理后，猪场废水中残留有机物成分在厌氧氨氧化反应过程中无显著变化，主要为酯类和烷烃类物质；残留有机物对厌氧氨氧化效果无明显影响。（5）采用PCR技术进行特殊功能菌种检测，结果表明模拟废水处理系统和猪场废水处理系统的菌群中均含有厌氧氨氧化菌和好氧硝化菌；通过blast比对，厌氧氨氧化菌扩增序列与未培养的Planctomycetales菌和Candidatus Brocadia fulgida菌16S rRNA部分序列相似性分别为95％、90%。（6）MPN法菌种计数结果显示，模拟废水处理系统和猪场废水处理系统的菌群中均含有硝化细菌、亚硝化细菌和少量反硝化菌，实验条件下的微生物系统是一个厌氧氨氧化菌与好氧硝化菌、反硝化菌共存的系统。 Poultry wastewater is one of the main source of water pollution in rural areas，and nitrogen removal is the most difficult part in treating poultry wastewater. There are some disadvantages in traditional nitrogen removal, such as high energy consumption and more additional organic carbon. It is important to develop ecolomical and efficient technologyies. Shortcut nitricfication/denitrification, as a pretreatment process, was combined with Anammox in this research, so that part of total nitrogen and most degradable COD could be removed by the former, and further nitrogen removal could be implemented by the latter. The combination of the two technologies was a new approach to treat wastewater with high ammonium and low C/N. Piggery wastewater with low C/N was treated in lab-scale experiment. Firstly, shortcut nitrification/denitrification was investigated, and Anammox reactor was started up successfully at the same time. Then piggery wastewater after pretreatment was treated by Anammox. The results showed :(1) It was feasible to take nitrification/denitrification as the pretreatment process of Anammox. By using this process, part of total nitrogen and COD were removed, the ratio of ammonium and nitrite reached around 1︰1 and the pH was about 7.8, which were favorable for Anammox. The average removal percentage of COD and total nitrogen were about 64.3% and 49.1%, respectively. (2) Simulated wastewater was used to start up Anammox reactor. The reactor was started up successfully within 5 months and stable performance was achieved. The highest nitrogen removal reached 87.1% and the biggest volumetric total nitrogen removal rate reached 0.14kg/m3.d. The average ratio of ammonium, nitrite and nitrate was 1:1.21:0.33. (3)Taking the effluent of shortcut nitrification/denitrification as the influent, the nitrogen removal efficiency of Anammox was stable, and the the average removal percentage of ammonium, nitrite, total nitrogen and COD were 93.0%, 99.4% , 84.6% and 18.1%, respectively, which had little difference with that by using simulated wastewater..(4) After pretreatment, the residual organic carbon in piggery wastewater showed no obvious change during the Anammox process, and the main organic compounds were saturated hydrocarbon and ester, which had no obvious negative effect on Anammox process.(5) By PCR technology, the existence of Anammox bacteria was confirmed and the aerobic nitrifying bacteria was found to coexist as well. The result of blast showed that the identities of Anammox bacterium to part of 16S rRNA sequence of uncultured Planctomycetales bacterium and Candidatus Brocadia fulgida bacterium were 95% and 90%, respectively.（6）By MPN method, nitrite oxidizer, ammonium oxidizer and denitrification bacteria were detected in both simulated and piggery wastewater treatment system of Anammox, and the microorganism system was composed of Anammox bacteria，aerobic bacteria and denitrification bacteria together.

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.

有机碳对混合菌群亚硝酸盐氮氧化的影响

自养硝化过程在自然界氮素循环和污水处理系统脱氮过程中起着关键作用。因此，了解有机碳对硝化的影响和硝化菌与异养菌之间的竞争对微生物生态学和污水处理系统设计都很重要。目前对氨氧化到硝酸盐氮过程的研究文献很多，但对亚硝酸盐氧化过程在异养菌的存在下如何受到有机碳影响的研究甚少。本文从生理生化指标、基因组学、蛋白组学三方面考察了在实验室条件下有机碳（乙酸钠）对硝化细菌和异养菌组成的混合菌群的硝化性能、菌群结构及代谢功能的变化的影响。 全文分为两大部分： 第一部分为乙酸钠对游离态硝化混合菌群的硝化性能和菌群结构的短期影响。混合菌株先在自养条件下进行连续培养，两个月后硝化速率达到20 mg N/(L·d)；而后离心收集菌体进行批式实验。在批式反应器中，初始亚硝氮均为126mg N/ L，乙酸钠-C 与亚硝酸盐-N 的比分别为0，0.44，0.88，4.41，8.82。结果表明：在低C/N 比（0.44 和0.88）时，亚硝酸盐去除速率比C/N=0 下高，细菌呈现一次生长；而在高C/N 比（4.41 和8.82）时，出现连续的硝化反硝化，亚硝酸盐去除率仍比对照下高，细菌呈现二次生长。不同C/N 比下微生物群落明显不同，优势菌群从自养和寡营养细菌体系（包括亚硝酸盐氧化菌，拟杆菌门,α-变形菌纲,浮霉菌门和绿色非硫细菌下的一些菌株）过渡到异养和反硝化菌体系 （γ-变形菌纲的菌株尤其是反硝化菌Pseudomonas stutzeri 和P. nitroreducens 占主导）。 第二部分为乙酸钠对硝化混合菌群生物膜的硝化性能和菌群结构的长期影响。接种富集的硝化混合菌群于装有组合式填料的三角瓶中，于摇床中自养培养；两个月后填料上形成生物膜的硝化速率达到20 mg N/ (L·d)；而后进行长期实验，每12 小时更换混合营养培养基（亚硝氮约200 mg N/ L，C/N 比同上）。结果显示：相较于C/N 比=0 时的亚硝酸盐氧化反应来说，低C/N 比出现了部分的反硝化，而高C/N 比则是几乎完全的反硝化。与对照比，C/N=0.44 时亚硝酸盐氧化速率并未受乙酸钠的影响，反而上升了，但C/N=0.88 时亚硝酸盐氧化速率有所下降。菌群结构分析表明自养对照与混合营养下微生物群落的不同；PCR-DGGE未检测出混合营养下硝化杆菌的存在，而显示异养菌尤其是反硝化菌的大量存 在。荧光定量PCR 结果表明随C/N 比上升，硝化杆菌数量从2.42 × 104 下降到1.34× 103 16S rRNA gene copies/ ng DNA，反硝化菌由0 增加至2.51 × 104 nosZgene copies/ ng DNA。SDS-PAGE 的结果表明不同C/N 比下的蛋白组较为复杂且呈现一定的差异性。 有机碳对亚硝氮氧化及微生物群落的影响很复杂，本文分别讨论了对游离态和生物膜固定态两种状态的混合菌群相应的短期和长期影响研究。研究发现，有机碳并非一定带来硝化的负影响，如果控制在适当的C/N 比范围，有机碳是有利于亚硝氮氧化的。这些发现阐明了有机碳和硝化反硝化的关系，填补了硝化微生物生态学上的空白，对污水处理系统中减少异养菌的影响并提高氮去除率有一定理论指导意义。 Nitrification plays a key role in the biological removal of nitrogen in both nature and wastewater treatment plant (WWTP). So, understanding of the effect of organic carbon on nitrification and the competition between nitrifying bacteria and heterotrophic bacteria is important for both microbial ecology and WWTP design and operation. Despite the fact that the nitrification process of ammonia to nitrate has been extensively investigated, it is not known how the process of nitrite oxidization is affected by organic carbon when heterotrophic bacteria are present. By measuring different physiological and biochemical parameters, as well as using genomic DNA and proteome analysis, we investigated the influence of organic (acetate) on nitrite oxidizing performance, community structure and metabolic function of nitrite-oxidizing and heterotrophic bacteria under laboratory conditions. The dissertation involves two parts: Part one deals with the effect of organic matter on functional performance and bacterial community shift of nitrite-oxidizing and heterotrophic bacteria under suspended state. The bacteria were prepared in a continuous-flow stirred reactor under autotrophic condition; after two months, the nitrification rate of the culture reached about 20 mg N/ (L·d); then the bacteria were harvested for the next batch experiments. The initial concentrations of nitrite were 126 ± 6 mg N/ L in all flasks, and sodium acetate (C) to nitrite (N) ratios were 0, 0.44, 0.88, 4.41, and 8.82, respectively. The results showed that at low C/N ratios (0.44 or 0.88), the nitrite removal rate was higher than that obtained under autotrophic condition and the bacteria had single growth phase, while at high C/N ratios (4.41 or 8.82), continuous aerobic nitrification and denitrification occurred besides higher nitrite removal rates, and the bacteria had double growth phases. The community structure of total bacteria strikingly varied with the different C/N ratios; the dominant populations shifted from autotrophic and oligotrophic bacteria (NOB, and some strains of Bacteroidetes, Alphaproteobacteria, Actinobacteria, and green nonsulfur bacteria) to heterotrophic and denitrifying bacteria (strains of Gammaproteobacteria, especially Pseudomonas stutzeri and P. nitroreducens). Part two describes the influence of acetate on nitrite oxidizing performance, community structure and metabolic function of nitrite-oxidizing and heterotrophic bacteria in biofilms. Bacterial enrichments was transferred into flasks with polypropylene carriers and cultured under agitated and autotrophic condition. After two month, the biofilms grown on the carriers had a nitrification rate of about 20 mg N/ (L·h); then the biofilms were refreshed with mixotrophic medium (nitrite were 200 mg N/ L in all flasks, and C/N ratios was the same as above) every 12 h. the results show: normal nitrite oxidization reactions were performed when C/N = 0, but nitrite oxidization and partial denitrification occurred with low C/N ratios (0.44 or 0.88). At high C/N ratios (4.41 or 8.82), we mainly observed denitrification. In contrast to C/N = 0, the nitrite oxidization rate was unaffected when C/N = 0.44, but decreased with C/N = 0.88. The structure of bacterial communities varied significantly between autotrophic and mixotrophic conditions. Nitrobacter was hard to detect by PCR-DGGE while heterotrophs and especially denitrifiers were in the majority under mixotrophic conditions. Real-time PCR indicated that the Nitrobacter population decreased from 2.42 × 104 to 1.34 × 103 16S rRNA gene copies/ ng DNA, while the quantity of denitrifiers obviously increased from 0 to 2.51×104 nosZ gene copies/ ng DNA with an increasing C/N ratio. SDS-PAGE indicated the complexity of and a certain difference between the proteome of nitrite-oxidizing and heterotrophic bacteria at different C/N ratios. We conclude that the influence of organic matter on nitrite oxidation and the community structure of NOB and heterotrophic bacteria is complex. In this dissertation, we focused on how sodium acetate influenced the system both under suspended state and in biofilms. We observed that acetate did not necessarily have a negative impact on nitrification. Instead, an appropriate amount of acetate benefited both nitrite oxidization and denitrification. These findings provide a greater understanding about the relationship between organics and nitrification; they fill the gaps in the field of microbial ecology of nitrifying bacteria; they also provide insight into how to minimize the negative impact of heterotrophic bacteria and maximize the benefit of nitrogen removal in biological treatment systems.

重离子诱变技术选育碱性蛋白酶高产菌株；The Selection for High-yield Alkaline Protease Producing Strain by Heavy-ion Irradiation

从采集的土壤样品中分离筛选出一株碱性蛋白酶产生菌G-41,经16S rRNA分子鉴定为芽孢杆菌属菌株。该菌株在发酵培养基中能产生较高产量的胞外碱性蛋白酶(1.7×104U/mL)。以G-41为出发菌株,对其进行重离子辐照诱变处理,获得突变株G-41-68,将该突变株再次经重离子诱变,从大量突变株中筛选出碱性蛋白酶高产菌株15Gy-54,其酶活力达到6.22×104U/mL。与出发菌株相比较,突变株G-41-68和15Gy-54的酶活力分别提高了1.58倍和2.65倍。对突变株15Gy-54的发酵条件进行了优化研究,结果表明,该菌株的碱性蛋白酶活力得到进一步提高,达到7.18×104U/mL,其最适发酵条件为:培养基(g/100mL)为胰蛋白胨1、酵母膏0.5、乳糖5、Na2HPO4·12H2O0.4、KH2PO40.03、Na2CO30.1、MgSO40.0481(4×10-3mol/L)、pH8.0,培养温度41℃,振荡培养时间42-48h。实验结果表明,重离子辐照诱变技术是一种非常有效的微生物诱变育种新技术。

镉胁迫对拟南芥幼苗错配修复基因表达的影响

采用半定量反转录聚合酶链式反应(RT-PCR)技术研究了Cd胁迫对拟南芥(Arabidopsis thaliana)幼苗错配修复和增殖细胞核抗原基因表达的影响,并结合幼苗的形态和生理指标,选取Cd胁迫敏感的生物标记物。结果表明:不同浓度(0.25、0.5、1.0 mg.L-1)Cd处理对拟南芥幼苗叶片数、地上部鲜质量影响不大;Cd浓度为0.25 mg.L-1时,地上部分可溶性蛋白质含量明显升高(P<0.05),Cd浓度为0.5和1.0 mg.L-1时,可溶性蛋白质含量明显降低(P<0.05);叶绿素含量随着Cd浓度的增加而微弱增加(P>0.05)。Cd浓度为0.25 mg.L-1时,以18S rRNA为内参照,PCNA1、PCNA2、MSH2、MSH3、MSH6、MSH7 6个基因均出现了诱导表达,当Cd浓度增加到1.0 mg.L-1时,除了MSH6持续表达诱导及MSH3基因与对照相比表达抑制外,其他基因的表达依然出现诱导,但都低于0.5 mg.L-1Cd处理下的基因表达水平。以上结果表明,基因表达的改变可作为检测Cd污染对植物遗传毒性效应潜在有用的生物标记物。

拮抗杉木致害菌Fusariumoxysporum海洋细菌3728的生物学特征及其鉴定

从南海红树林木榄(Bruguiera gymnorrhizo)根际土壤中分离到一株具有拮抗杉木致害菌尖孢镰刀菌萎蔫专化型SF2(Fusariumoxysporum f.sp.vasinfectum)活性的海洋细菌3728菌株,对分离菌株的形态特征、培养特征、生理生化特征和16S rRNA基因序列进行了系统的研究。发现细菌3728与枯草芽孢杆菌(Bacillus subtilis)序列相似性最高,达到100%,在系统进化树中与枯草芽孢杆菌(Bacillus subtilis AJ276351)处于同一分支上,结合形态和生理生化分析结果,将其鉴定为枯草芽孢杆菌。

Soil microbial community analysis using denaturing gradient gel electrophoresis

The most biological diversity on this planet is probably harbored in soils. Understanding the diversity and function of the microbiological component of soil poses great challenges that are being overcome by the application of molecular biological approaches. This review covers one of many approaches being used: separation of polymerase chain reaction (PCR) amplicons using denaturing gradient gel electrophoresis (DGGE). Extraction of nucleic acids directly from soils allows the examination of a community without the limitation posed by cultivation. Polymerase chain reaction provides a means to increase the numbers of a target for its detection on gels. Using the rRNA genes as a target for PCR provides phylogenetic information on populations comprising communities. Fingerprints produced by this method have allowed spatial and temporal comparisons of soil communities within and between locations or among treatments. Numerous samples can be compared because of the rapid high throughput nature of this method. Scientists now have the means to begin addressing complex ecological questions about the spatial, temporal, and nutritional interactions faced by microbes in the soil environment.

Novel uncultivated labyrinthulomycetes revealed by 18S rDNA sequences from seawater and sediment samples

Labyrinthulomycetes (Labyrinthulea) are ubiquitous marine osmoheterotrophic protists that appear to be important in decomposition of both allochthonous and autochthonous organic matter. We used a cultivation-independent method based on the labyrinthulomycete-specific primer LABY-Y to PCR amplify, clone, and sequence 68 nearly full-length 18S rDNA amplicons from 4 sediment and 3 seawater samples collected in estuarine habitats around Long Island, New York, USA. Phylogenetic analyses revealed that all 68 amplicons belonged to the Labyrinthulea. Only 15 of the 68 amplicons belonged to the thraustochytrid phylogenetic group (Thraustochytriidae). None of these 15 were similar to cultivated strains, and 11 formed a novel group. The remaining 53 amplicons belonged either to the labyrinthulid phylogenetic group (Labyrinthulidae) or to other families of Labyrinthulea. that have not yet been described. Of these amplicons, 37 were closely related to previously cultivated Aplanochytrium spp. and Oblongichytrium spp. Members of these 2 genera were also cultivated from 1 of the sediment samples. The 16 other amplicons were not closely related to cultivated strains, and 15 belonged to 5 groups of apparently novel labyrinthulomycetes. Most of the novel groups of amplicons also contained environmental sequences from surveys of protist diversity using universal 18S rDNA primers. Because the primer LABY-Y is biased against several groups of labyrinthulomycetes, particularly among the thraustochytrids, these results may underestimate the undiscovered diversity of labyrinthulomycetes.

Differences in the rDNA-bearing chromosome divide the Asian-Pacific and Atlantic species of Crassostrea (Bivalvia, Mollusca)

Karyotype and chromosomal location of the major ribosomal RNA genes (rDNA) were studied using fluorescence in situ hybridization (FISH) in five species of Crassostrea: three Asian-Pacific species (C. gigas, C. plicatula, and C. ariakensis) and two Atlantic species (C. virginica and C. rhizophorae). FISH probes were made by PCR amplification of the intergenic transcribed spacer between the 18S and 5.8S rRNA genes, and labeled with digoxigenin-11-dUTP. All five species had a haploid number of 10 chromosomes. The Atlantic species had 1-2 submetacentric chromosomes, while the three Pacific species had none. FISH with metaphase chromosomes detected a single telomeric locus for rDNA in all five species without any variation. In all three Pacific species, rDNA was located on the long arm of Chromosome 10 (10q)-the smallest chromosome. In the two Atlantic species, rDNA was located on the short arm of Chromosome 2 (2p)-the second longest chromosome. A review of other studies reveals the same distribution of NOR sites (putative rDNA loci) in three other species: on 10q in C. sikamea and C. angulata from the Pacific Ocean and on 2p in C. gasar from the western Atlantic. All data support the conclusion that differences in size and shape of the rDNA-bearing chromosome represent a major divide between Asian-Pacific and Atlantic species of Crassostrea. This finding suggests that chromosomal divergence can occur under seemingly conserved karyotypes and may play a role in reproductive isolation and speciation.

Particle-attached and free-living bacterial communities in a contrasting marine environment: Victoria Harbor, Hong Kong

Diversity of particle-attached and free-living marine bacteria in Victoria Harbor, Hong Kong, and its adjacent coastal and estuarial environments was investigated using DNA fingerprinting and clone library analysis. Denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA genes showed that bacterial communities in three stations of Victoria Harbor were similar, but differed from those in adjacent coastal and estuarine stations. Particle-attached and free-living bacterial community composition differed in the Victoria Harbor area. DNA sequencing of 28 bands from DGGE gel showed Alphaproteobacteria was the most abundant group, followed by the Bacteroidetes, and other Proteobacteria. Bacterial species richness (number of DGGE bands) differed among stations and populations (particle-attached and free-living; bottom and surface). BIOENV analysis indicated that the concentrations of suspended solids were the major contributing parameter for the spatial variation of total bacterial community structure. Samples from representative stations were selected for clone library (548 clones) construction and their phylogenetic distributions were similar to those of sequences from DGGE. Approximately 80% of clones were affiliated to Proteobacteria, Bacteroidetes and Cyanobacteria. The possible influences of dynamic pollution and hydrological conditions in the Victoria Harbor area on the particle-attached and free-living bacterial community structures were discussed.

Particle-attached and free-living bacterial communities in a contrasting marine environment: Victoria Harbor, Hong Kong

Diversity of particle-attached and free-living marine bacteria in Victoria Harbor, Hong Kong, and its adjacent coastal and estuarial environments was investigated using DNA fingerprinting and clone library analysis. Denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA genes showed that bacterial communities in three stations of Victoria Harbor were similar, but differed from those in adjacent coastal and estuarine stations. Particle-attached and free-living bacterial community composition differed in the Victoria Harbor area. DNA sequencing of 28 bands from DGGE gel showed Alphaproteobacteria was the most abundant group, followed by the Bacteroidetes, and other Proteobacteria. Bacterial species richness (number of DGGE bands) differed among stations and populations (particle-attached and free-living; bottom and surface). BIOENV analysis indicated that the concentrations of suspended solids were the major contributing parameter for the spatial variation of total bacterial community structure. Samples from representative stations were selected for clone library (548 clones) construction and their phylogenetic distributions were similar to those of sequences from DGGE. Approximately 80% of clones were affiliated to Proteobacteria, Bacteroidetes and Cyanobacteria. The possible influences of dynamic pollution and hydrological conditions in the Victoria Harbor area on the particle-attached and free-living bacterial community structures were discussed.

Genome-wide survey of putative serine/threonine protein kinases in cyanobacteria

Background: Serine/threonine kinases (STKs) have been found in an increasing number of prokaryotes, showing important roles in signal transduction that supplement the well known role of two-component system. Cyanobacteria are photoautotrophic prokaryotes able to grow in a wide range of ecological environments, and their signal transduction systems are important in adaptation to the environment. Sequence information from several cyanobacterial genomes offers a unique opportunity to conduct a comprehensive comparative analysis of this kinase family. In this study, we extracted information regarding Ser/Thr kinases from 21 species of sequenced cyanobacteria and investigated their diversity, conservation, domain structure, and evolution. Results: 286 putative STK homologues were identified. STKs are absent in four Prochlorococcus strains and one marine Synechococcus strain and abundant in filamentous nitrogen-fixing cyanobacteria. Motifs and invariant amino acids typical in eukaryotic STKs were conserved well in these proteins, and six more cyanobacteria- or bacteria-specific conserved residues were found. These STK proteins were classified into three major families according to their domain structures. Fourteen types and a total of 131 additional domains were identified, some of which are reported to participate in the recognition of signals or substrates. Cyanobacterial STKs show rather complicated phylogenetic relationships that correspond poorly with phylogenies based on 16S rRNA and those based on additional domains. Conclusion: The number of STK genes in different cyanobacteria is the result of the genome size, ecophysiology, and physiological properties of the organism. Similar conserved motifs and amino acids indicate that cyanobacterial STKs make use of a similar catalytic mechanism as eukaryotic STKs. Gene gain-and-loss is significant during STK evolution, along with domain shuffling and insertion. This study has established an overall framework of sequence-structure-function interactions for the STK gene family, which may facilitate further studies of the role of STKs in various organisms.