Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences
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表面活性剂及其纳米自组装结构对甲烷水合物生成的作用
No full text天然气水合物是天然气分子和水分子在一定温度、压力条件下（如，3 MPa、275.15 K）形成的类冰的、笼型结晶化合物，天然气水合物因储气量高（180 v/v）、储存条件温和而被视为极具应用潜力的天然气储运技术，如何实现天然气水合物的快速生成是利用该技术的关键。
表面活性剂已被证实可以有效促进天然气水合物的生成，这极大地提高了水合物法储运天然气技术工程化的可行性，过去二十年中人们对表面活性剂在天然气水合物中的应用进行了大量研究，但至今仍面临一些难题。一方面，尽管表面活性剂对天然气水合物生成具有明显的促进作用，但其促进机理至今仍没有合理完善的解释；另一方面，在表面活性剂这一庞大的化合物体系中只有阴离子表面活性剂（如，十二烷基硫酸钠）对天然气水合物生成具有较为有效的促进作用，而其他类型表面活性剂的促进效果比较差；此外，以表面活性剂为促进剂时水合物分解过程中会产生大量泡沫，这不仅导致表面活性剂的流失，而且极不利于水合物在天然气储运中的应用。鉴于上述表面活性剂在天然气水合物应用中的缺陷，本论文以甲烷水合物为研究对象，首先对表面活性剂对水合物生成的促进机理进行系统研究，然后将表面活性剂在纳米尺度进行自组装以改善其在天然气水合物中的应用性能。本论文主要研究内容和结果如下：
（1）以具有相同碳链和不同亲水性基团的阴离子表面活性剂十二烷基磺酸钠（SDSN）、十二烷基硫酸钠（SDS）、十二烷基苯磺酸钠（SDBS）为促进剂研究阴离子表面活性剂对甲烷水合物生成的促进机理。结果表明，以SDSN和SDS为促进剂时，水合物沿反应釜内壁快速向上生长，生长过程在30 min内完成，这是因为SDSN和SDS可以明显改善反应液在反应釜内壁的润湿性，从而促使水合物沿反应釜内壁向上生长，使反应液在毛细效应作用下不断被吸到水合物表面以维持持续的气液接触，从而促使水合物快速生长；而以SDBS为促进剂时，水合物主要在反应釜底部生成，生长阶段持续数小时之多，这一方面是因为SDBS通过胶束作用促进水合物生成，水合物以胶束为核在反应液相中形成，另一方面是因为SDBS溶液在反应釜内壁的润湿性较差，不利于水合物沿反应釜内壁向上生长，因此，当在反应釜内安装亲水性较好的玻璃内衬后SDBS体系中水合物也出现明显向上生长的趋势。
（2）以水合物条件下不能形成胶束SDS为促进剂、在具有不同亲疏水性内壁的反应釜内进行甲烷水合物实验，进一步研究反应釜内壁的亲疏水性（反应液在反应釜内壁的润湿性）对甲烷水合物生长模式的作用，然后通过局部改变反应釜内壁的亲疏水性实现水合物生长方向的可控。结果表明，在亲水性较好的硅酸盐玻璃反应釜内甲烷水合物主要沿反应釜内壁向上生长，而在疏水性较好的PS塑料反应釜内水合物主要在反应釜底部生成；将玻璃反应釜内壁进行疏水化处理可以有效抑制水合物向上的生长；将反应液固定在反应釜中间位置时，水合物同时向上、向下生长，当将反应釜上部分进行疏水化处理后，水合物主要向下生长，但是，将反应釜下部分进行疏水化处理后，水合物主要向上生长。
（3）在具有较好亲水性的玻璃反应釜内、以水合物条件下不能形成胶束的十二烷基硫酸钠（SDS）和能形成胶束的十二烷基苯磺酸钠（SDBS）为促进剂进行甲烷水合物实验，进一步研究表面活性剂胶束对甲烷水合物生长模式的作用。结果发现，以SDS为促进剂时水合物沿反应釜内壁向上生长而以SDBS为促进剂时水合物在反应釜底部生成；当SDS以胶束状结构存在于反应体系中时，水合物主要在反应釜底部生成，当SDBS以非胶束态存在于反应体系中时，水合物沿反应釜内壁向上生长。
（4）以乳液聚合法将表面活性剂固定在聚苯乙烯纳米球表面以实现表面活性剂在纳米尺度的固定化组装，研究表面活性剂的固定化组装在甲烷水合物生成中的作用。结果表明，水合物首先在固定表面活性剂的纳米球表面成核，然后以纳米球为核在反应液相中形成；在水合物生成过程中表面活性剂的固定化组装可以明显缩短水合物生成的诱导期、提高水合物的反应速率，同时提高形成的水合物的表观密度和储气量；在水合物分解过程中，阴离子、阳离子、非离子表面活性剂的固定化组装使甲烷回收率分别由76.37 %、 27.87 %、 32.35 %提高至87.55 %、43.43 %、60.20 %，并且能有效抑制泡沫的产生，此外，固定的表面活性剂表现出良好的循环利用性能。
（5）将阳离子表面活性剂十六烷基三甲基溴化铵CTAB以静电作用接在具有π-π共轭结构的活性红-195染料分子上，利用π-π共轭分子间的强作用力实现表面活性剂分子稳定的自组装（命名为CTAB@RR195），并将这种自组装结构体应用于甲烷水合物实验。结果发现，CTAB在RR195作用下自组装成尺寸为50-100 nm的多孔立方体结构，并且CTAB@RR195结构体对甲烷水合物生成的促进作用明显优于同浓度的CTAB，CTAB@RR195不但能更好的促进甲烷水合物的成核，还能使水合物生长阶段从数十小时缩短至1-2小时。Natural gas hydrates are ice-like crystalline compounds formed by gas and water molecules at suitable temperature and pressure (e.g. 3 MPa and 275.15 K), which are of significant potential in natural gas storage and transportation due to the high storage capacity (180 v/v) and mild storage condition. How to achieve rapid hydrate formation is critical to utilize hydrate-based natural gas storage and transportation.
Surfactants have been confirmed to effectively promote gas hydrate formation, which makes the hydrate-based natural gas storage and transportation more feasible. During the past two decades, lots of research has been carried out on the application of surfactants in gas hydrate formation, however, there are still several problems. On one hand, the promotion mechanism of surfactants to gas hydrate formation still remains poorly understood; on the other hand, even many kinds of surfactants have been used as promoters in gas hydrate formation, only anionic ones, especially sodium dodecyl sulfate, show the most efficient promotion, while other kinds of surfactants produce obviously less efficient promotion; in addition, the existence of surfactants leads to a lot of foam generated during the hydrate dissociation, which not only causes the outflow of surfactants but impacts the application of hydrates. Given the defects above, in this work, the promotion mechanism of surfactants in methane hydrate formation was systematicly studied, and then surfactants were assembled at nano-scale to improve the application performance in gas hydrate formation.
(1) Three anionic surfactants with the same carbon chain and different head groups, sodium dodecyl sulfonate (SDSN), sodium dodecyl sulfate (SDS) and sodium dodecyl benzene sulfonate (SDBS) were used to study the promotion mechanism of anionic surfactants to gas hydrate formation. When SDSN and SDS were used, hydrates formed on the reactor sidewall and grew upwards and the hydrate growth period was finished within 30 min. This was because SDSN and SDS could improve the wettability of reaction solution on the reactor sidewall and lead to the hydrates grow on the reactor sidewall, then reaction solution was sucked under capillary effect to the hydrate surface to keep continuous liquid-gas contact, resulting in rapid hydrate growth. When SDBS was used, hydrates formed in the bottom of the reactor. One possible reason was that SDBS promoted gas hydrate formation by micelle effect, and hydrates were formed with SDBS micelles as nuclei in the reaction phase. Another reason was that SDBS led to poor wettability of reaction solution on the reactor sidewall, which was not conducive to the upward hydrate growth, therefore, when a glass layer was installed in the reactor obvious upward hydrate growt was observed in SDBS system.
(2) Methane hydrate formation was carried out with SDS that could not form micelles under hydrate condition as promoter in reactors with different surface hydrophilicity-hydrophobicity properties to study the effects of reactor surface property on gas hydrate growth pattern, and then direction controlled gas hydrate growth was achieved through partly changing the surface hydrophilicity- hydrophobicity of the reactor sidewall. In the glass reactor with good hydrophilicity hydrates grew upwards on the sidewall, while in the plastic reactor with good hydrophobicity hydrates formed in the bottom of the reactor. When the glass reactor was hydrophobicly modified, the upward hydrate growth was obviously hindered. When reaction solution was fixed in the middle of reactor hydrates grew both upwards and downwards. When the upper part of the reactor was hydrophobicly modified hydrates grew downwards, while when the lower part of the reactor was hydrophobicly modified hydrates grew upwards.
(3) Methane hydrate formation was carried out in glass reactors with surfactants could and could not form micelles at hydrate formation temperature to study the effects of surfactant micelles on methane hydrate growth pattern, which were sodium dodecyl benzene sulfonate (SDBS) and sodium dodecyl sulfate (SDS). When SDS was used hydrates grew upwards on the reactor sidewall, while when SDBS was used hydrates formed in the bottom of the reactor. When SDS-coated polystyrene nanospheres were used, SDS existed in the reaction solution in the form of mimic micelles and consequently much less extent of upward hydrate growth was achieved. When SDBS was used together with SDS at non-micelle forming condition, prominent upward hydrate growth was obtained.
(4) Surfactants were fixed on the surface of polystyrene nanospheres through emulsion polymerization to achieve the assembly of surfactants at nanoscale, then the effects of fixation of surfactants in gas hydrate formation was studied. In methane hydrate formation with nanospheres, hydrates nucleated firstly on the surface of the nanospheres and then formed with nanospheres as nuclei in the liquid phase. During gas hydrate formation, the fixation of surfactants could reduce the induction periods and improve the apparent densities and storage capacities of the formed hydrates. During hydrate dissociation, the fixation of anionic, cationic and nonionic surfactants could improve the methane recoveries from 76.37 %, 27.87 %, and 32.35 % to 87.55 %, 43.43 %, and 60.20 % respectively and impeded the foam generation efficiently. In addition, the fixed surfactants produced very good recycling performance in gas hydrate formation.
(5) Cetyltrimethyl ammonium bromide (CTAB) was fixed on π-π conjugate reactive red 195 molecules through electrostatic interaction, which was then applied in methane hydrate formation. As a result, porous nanostructures with the size of 50-100 nm were obtained through the self-assembly of CTAB with RR195 (named as CTAB@RR195). CTAB@RR195 produced much better promotion to methane hydrate formation compared to CTAB with the same concentration. CTAB@RR195 could not only promote hydrate nucleation, but also significantly reduce the hydrate growth period from dozens of hours to one or two hours.
Keywords: Methane hydrates, Surfactants, Self-assembled nanostructures, Promotion mechanis
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No full text微生物代谢产物可以分为胞外产物和胞内产物两类。与胞外产物相比，胞内产物的分离首先要通过物理或化学手段进行细胞破碎，使代谢产物释放。目前常用的细胞破碎方法存在着能耗高、污染大等弊端，因此我们希望构建一个自诱导裂解系统，使细胞在发酵后期裂解并释放产物。
大肠杆菌λ噬菌体裂解基因（SRRZ）编码三个蛋白，其中基因S编码穿孔素（holin），攻击内膜蛋白；基因R编码裂解素（endolysin），使细胞壁水解；基因RZ编码辅助裂解因子。将裂解基因连接到质粒pBAD18上表达，构建了重组菌株Q2537。在Q2537培养过程加入阿拉伯糖诱导后，菌体迅速裂解，证明了裂解基因的有效性。考虑到实际生产过程，我们希望建立一种更为经济、简单的裂解系统。
基因mgtA的转录受到二组分系统PhoPQ和其mRNA 5’非翻译区（5’UTR）内核糖开关的共同控制。Mg2+浓度较低的条件会激活受PhoP调节基因mgtA的表达；在Mg2+浓度较高的条件下，基因mgtA的表达受到抑制。mgtA核糖开关可作为细胞内Mg2+浓度传感器，并根据Mg2+浓度形成不同的高级结构，当细胞内Mg2+浓度升高时，核糖开关的特定结构会在转录进行到编码区之前将其提前终止。
在本研究中，我们克隆了λ噬菌体裂解基因SRRZ，对裂解基因的裂解效果进行了验证，发现在1小时内，菌株即表现出明显的裂解效果；然后在裂解基因前插入基因mgtA的启动子PmgtA. 重组质粒在不同宿主中均表现出明显的裂解效果，裂解效率在90%以上，但是在聚3-羟基丙酸（P3HP）的发酵过程中发现，质粒丢失现象严重，产物释放率较低。因此，我们将片段PmgtA-SRRZ克隆到P3HP生产菌株Q1638携带的质粒pW01中，使双质粒系统转变为单质粒系统，质粒稳定性提高2.7倍，同时在聚3-羟基丙酸（P3HP）的发酵过程中，P3HP的产量占细胞干重的70 %以上，比原始菌株提高了2.37倍，单质粒裂解系统产物释放率大幅提高；为进一步提高基因的稳定性，同时减少抗生素的使用，降低生产成本，我们利用染色体整合（Chromosome Gene Integration，CGI）手段，将裂解系统的基因片段整合到基因组染色体上进行表达，但是CGI裂解效果较差，将启动子更换为PT7或Plac后，短时间内仍无明显的裂解效果。
随着现代生物技术手段的不断发展，微生物法合成生物化工产品得到了迅速地发展。经济方便的产品分离提纯过程，对微生物发酵的发展十分重要。我们建立的肠杆菌自诱导裂解系统，可以通过微生物代谢过程中对Mg2+的消耗，在发酵后期自行裂解并释放产物，操作方便；裂解基因的表达不需要使用昂贵的诱导剂，经济性高；同时，裂解系统的裂解效率高，产物释放率高。Mg2+诱导的大肠杆菌自诱导裂解系统除用于P3HP的产物回收外，还可用于其他胞内产物如蛋白质等的分离，为微生物代谢产物的分离提供了新思路，对微生物发酵产业的发展意义深远。Microbial metabolites can be divided into two categories, extracellular products and intracellular products. Compared with the extracellular products, the separation of intracellular products is more complicated. First the cells should be broken up by physical or chemical methods to release the metabolites. The common methods of cell disruption has big drawbacks such as high energy consumption, high pollution. So we want to build an auto-inducible lysis system which can make the cells break up during the later period of fermentation.
λ bacteriophage lysis genes (SRRZ) has three subunits, gene S encodes the protein of holin and holin accumulates in the cell membrane, gene R encodes the protein of endolysin and endolysin hydrolyzes the cell wall, gene RZ encodes the cofactor. The lysis genes were inserted into the plasmid pBAD18 and the recombinant strains Q2537 was constructed. In the process of the culture of Q2537, arabinose was added to induce the lysis genes expression. The cell concentration dropped quickly by induction. And considering the practical production proces, we hope to establish a more economic and simple lysis system.
It is suggested that two independent mechanisms are involved in Mg2+-dependent transcriptional regulation of mgtA. (i) The PhoP/PhoQ two-component system responds to micromolar levels of environmental Mg2+ and activates transcription initiation; or to milli-molar levels of Mg2+ and represses transcription initiation. (ii) Once transcription is initiated, the 50 untranslated region of nascent mgtA transcripts functions as an alternative Mg2+-sensing system. If the Mg2+ concentration increases in the bacterial cytoplasm, the latter system interrupts mgtA transcription before it is extended to the downstream coding region.
In this study, λ bacteriophagelysis genes (SRRZ) was cloned, and then the promter of mgtA was inserted before SRRZ. Because the promter PmgtA will activate genes expression in milli-molar levels of Mg2+ and more Mg2+ will be applied to keep the regular growth of cells which will adversely affect the accumulation of metabolites. So the 5' untranslated region (5'-UTR) of mgtA and the promter PmgtA were both inserted to ensure SRRZ express in micromolar levels of environmental Mg2+. To improve the plasmid stability, genetic fragments PmgtA and PmgtA-UTR were cloned into the plasmid PW01 respectively and the double plasmid system was changed into single plasmid system. In order to further increase the stability of the genes and reduce antibiotic use simultaneously, the Mg2+-inducible lysis system was integrated into the host genome using chromosome gene integration（CGI） method.
In lysis capacity experiments, the lysis rates of all the recombinant strains except CGI strains were all more than 90%. The plasmid stability was 2.7 times higher in single plasmid system than the double plasmid system. P3HP was accounted for 50% of total dried cell，which was 2.37 times higher than the original strains. But the CGI strains did not show obviouslysis capacity.
With the development of modern bio-technology, microbial synthesis of bio-chemicalproducts developed rapidly. Aeconomical convenience method is very important for the developmentof microbial fermentation. The auto-inducible lysis system can make cells break up during the later period of fermentation by microbial metabolism of Mg2+ and it waseasy to operate; the expression of lysis genes did not need expensive inducer and the cracking efficiency and products release rate were high. The auto-inducible Escherichia coli lysis system can be applied on other products separation such as extracellular proteins. It provided new ideas to the isolation of metabolism products which has important significance to the development of animalcule fermentation engineering
聚球藻UTEX 2973 高光适应相关基因筛选及其糖类化合物合成潜力研究
No full text蓝细菌能够进行光合作用，是研究植物中光合及抗逆等生理过程机理的重要模式微生物，具有重要的理论研究意义。生物能源为可再生能源，是化石类能源的替代品之一，然而如何高效率、低成本地获取糖原料，是限制生物能源发展和应用的瓶颈问题之一。蓝细菌可以进行光合作用，并且具有生长速度快、遗传操作简便等优势，是生物能源生产中理想的细胞工厂。 聚球藻UTEX 2973 是目前报道的生长速度最快的蓝细菌，具有高温、高光耐受能力，而聚球藻PCC 7942 的高光、高温耐受性稍差。考虑到聚球藻UTEX 2973 与聚球藻PCC 7942 的基因组仅存在0.2%的差异，通过研究聚球藻UTEX 2973 与聚球藻PCC7942 的差异基因，我们尝试对高光耐受相关的基因进行鉴定；另外，对聚球藻UTEX 2973 合成糖类化合物的能力进行初步的探索。
本研究的主要内容及结果如下：
（1）通过将聚球藻UTEX 2973 与聚球藻PCC 7942 存在于基因中的55 个
SNPs 位点互补到聚球藻PCC 7942 中进行高光筛选，鉴定了聚球藻UTEX 2973中与高光适应相关的基因ATP synthase FoF1 subunit alpha，将聚球藻PCC 7942中该基因突变为聚球藻UTEX 2973 中的序列后，得到的聚球藻PCC 7942 突变株也具有了一定的高光耐受能力，证明ATP synthase FoF1 确实是与高光耐受相关的重要基因；
（2）通过对聚球藻UTEX 2973 糖原积累的检测，发现在正常BG11 培养下糖原积累量达到其细胞干重的51%，并且积累糖原的同时其生物质也迅速积累；
（3）聚球藻UTEX 2973 与聚球藻PCC 7942 有相似的耐盐能力，在不同盐浓度下的蔗糖生产能力也较为类似；将蔗糖转运蛋白CscB 在聚球藻UTEX 2973中过表达，成功实现了胞内蔗糖的外运并且得到的最高蔗糖产量3.34 g L-1；
（4）相同浓度的KCl 与NaCl 在胁迫聚球藻UTEX 2973 生产蔗糖上具有相
似的能力，但是KCl 对藻细胞产生的损伤较小，使得使用KCl 作为胁迫物质时可以更好的生产蔗糖。使用含有KCl 的培养基以半连续的方式培养聚球藻UTEX2973，在培养21 天时共积累了8.7 g L-1 的蔗糖，同时蓝细菌胞内糖原维持在干重的40%左右。Cyanobacteria are a group of prokaryotes that perform oxygenic photosynthesis, and were seen as research models in studying mechanisms in plants, such as
photosynthesis and stress resistance. Biofuel was one of the promising substitutes for fossil fuel, however the lack of efficiency and economic accesses to sugar feedstock is one of the bottleneck problems impeding the development and commercialization of biofuels. Besides the ability of photosynthesis, cyanobacteira have faster growth speed and easier genetic operation systems, and thus draw much attention recently both in the field of academic and industry.
Synechococcus elongatus UTEX 2973 is the fastest growing cyanobacterium reported up to now, whose genome sequence is 99.8% identical to that of
Synechococcus elongatus PCC 7942. Synechococcus UTEX 2973 has better tolerance to high light and high temperature than Synechococcus PCC 7942. Thus in this work, we tried to identify the high light tolerance related genes in Synechococcus UTEX 2973 based on the differences between two Synechococcus genomes, and explored the potential of Synechococcus UTEX 2973 for sugar feedstock production.
The main research contents and results are summarized as follows:
Firstly, the atpA gene encoding ATP synthase FoF1 subunit alpha was found to be responsible for high light acclimation in Synechococcus UTEX 2973 FoF1 through gene complementary experiments. The resulting Synechococcus PCC 7942 mutant
harboring the same single nucleotide polymorphism site with Synechococcus UTEX 2973 possesses tolerance to high light condition, which proved that ATP synthase FoF1 was involved in the high-light acclimation of Synechococcus UTEX 2973.
Secondly, Synechococcus UTEX 2973 accumulated glycogen of up to 51% of dry cell weight (DCW) during its fast accumulation of biomass under nitrogen replete
condition. Thirdly, Synechococcus UTEX 2973 possesses similar ability of salt tolerance, and has similar sucrose accumulation level under salt stress. Synechococcus UTEX 2973 can secrete sucrose into medium after overexpression of a sucrose transporter CscB. And a highest extracellular sucrose accumulation of 3.34 g L-1 was achieved in the CscB-expressing mutant of Synechococcus UTEX 2973. Finally, we found that KCl displays the same ability to induce sucrose production in Synechococcus UTEX 2973 as NaCl. However, KCl causes much lower damage to cells comparing with NaCl, which makes KCl a better choice for sucrose production.
Semi-continuous cultivation using medium containing KCl was performed, 8.7 g L-1 of sucrose was accumulated after 21 days of cultivation, and glycogen was maintained at 40% of DCW
蓝细菌生理与代谢工程新策略研究
No full text蓝细菌是重要的光合自养微生物，能够利用二氧化碳和太阳能合成有机物并释放氧气，也是最具潜力的生物燃料和生物基化学品光合平台，通过修饰蓝细菌天然代谢途径或向蓝细菌中引入人工构建的代谢途径已经实现了多种化合物的光合合成。乙醇是最具产业化潜力的生物燃料产品，也是最早报道、最具代表性的蓝细菌光合生物基化合物，以乙醇光合为模式进行蓝细菌生理和代谢工程策略的研究具有重要的示范意义。本研究从代谢途径和光合平台两个角度分别开展，探索开发新的优化策略和设计原则。
针对蓝细菌光合细胞工厂中乙醇合成的核心代谢途径PDCzm-slr1192，我们系统运用了体外重构和代谢工程两种策略对该途径进行深入、全面的动态解析。在代谢途径体外重构的基础上，我们系统评价了催化过程各种组分对反应速率的影响发现PDCzm的含量和活性是乙醇合成速率的限制因素，PDCzm和slr1192的最优浓度比为4:6，提高NADPH和丙酮酸的供应也应该是优化乙醇合成速率的重要方向。对体外重构和定量分析的结果，我们通过对蓝细菌的代谢工程改造加以验证。该部分研究为蓝细菌乙醇光合细胞工厂的下一阶段改造明确了方向，也为代谢途径的解析与优化提供了新的研究模式。
针对蓝细菌光合平台本身的生理和代谢特性与机制，我们主要进行糖原代谢系统进行改造和分析。与传统的阻断糖原合成的策略不同，我们采取过量表达糖原磷酸化酶来加速糖原降解的策略。结果我们发现糖原降解速率的加快，不但没有降低胞内糖原含量，反而使之大幅度提高，其原因是糖原磷酸化降解过程的加速降低了胞内磷酸的含量，别构激活了糖原合成的关键酶GlgC，从而使糖原合成得到加强，提高了糖原积累。在乙醇光合的蓝细菌工程藻株中，糖原磷酸化酶的过量表达也提高了糖原的积累，但同时乙醇产量和碳流分配比也得到提高；在限氮条件下，乙醇产量相对野生型藻株提高50%，证明糖原积累与代谢产物合成并非简单的碳流竞争关系。
我们的结果为蓝细菌生理与代谢工程研究提供了新的策略，为蓝细菌生理和代谢功能与调控机制研究提供了新的认识，将有助于开发更高效的蓝细菌光合平台。Cyanobacteria were a group of important photoautotrophic microorganisms, converting CO2 and solar energy into organic compounds through photosynthesis and releasing O2. Photosynthetic production of diverse chemicals in cyanobacteria has been achieved by modification of natural metabolic pathways or introduction of artificial metabolic pathways. Ethanol was the most promising biofuel product, the first reported and the most representative cyanobacteria based biochemical. Development of novel strategies for physiological and metabolic engineering in cyanobacteria is of great significance. This project aimed to explore new strategy and designing principles for optimization of metabolic pathways and photosynthetic platforms.
We adopted a combinatory strategy to understand and characterize the PDCzm-slr1192 pathway, essential for ethanol synthesis in cyanobacteria cell factories, by in vitro reconstitution and metabolic engineering. Based on the in vitro reconstitution system, we assessed the contributions of diverse components during the catalytic processes, and discovered that it was PDCzm rather than slr1192 holding control over the ethanol synthesis process, an optimal ratio of PDCzm-slr1192 was 4:6, and increasing the supply of NADPH and pyruvate should be the effective strategy for enhancing ethanol photosynthetic production. We performed metabolic engineering in cyanobacteria to confirm the hypothesis from in vitro reconstitution. The results provided a novel paradigm for characterizing metabolic pathways in cyanobacteria and inspired the direction for optimizing advanced ethanol photosynthetic production.
For understanding and optimizing physiological and metabolic characteristics of cyanobacteria cells, we modified and analyzed the system of glycogen metabolism. Comparing with the traditional glycogen synthesis blocking strategy, we overexpresses glycogen phosphorylase to accelerate the glycogen digestion processes. We discovered that enhanced glycogen phosphorylase activities did not decrease but significantly increase glycogen accumulations. Further analysis revealed that enhanced glycogen phosphorylase caused an intracellular phosphate-deprivation status, allosterically activated GlgC, and finally enhanced glycogen synthesis and accumulations. In engineered cyanobacteria strain for ethanol synthesis, overexpression of glgP simultaneously enhanced carbon partitioning ratio to glycogen and ethanol; under nitrogen deprivation conditions, ethanol synthesis was improved by 1.5-fold comparing with the wildtype strain, indicating glycogen accumulation was not a simple carbon flow competitive pathway with metabolite synthesis.
Our results demonstrated new strategies for physiological and metabolic engineering in cyanobacteria, and provided new understanding for function and regulation of metabolism and physiology, which would enable the development of more efficient photosynthetic platforms
高电压全固态锂离子电池界面问题及改性
No full text发展高电压全固态锂离子电池是提升锂离子电池能量密度和安全性的重要研究思路。但是，全固态锂离子电池中的固固界面阻抗较大，阻碍了锂离子在电极和电解质界面处的快速传输，而且正极材料的高工作电压容易引发电极和电解质之间的界面副反应，不利于电池循环寿命的提高。因此，高电压全固态锂离子电池面临极化较大、库伦效率较低、容量衰减迅速等问题。本论文以高电压全固态锂离子电池LiCoO2/PEO/Li为研究对象，分析了在高电压工作时（> 4.3 V）发生的界面副反应及其对电池性能的影响，提出了改善界面化学稳定性和提高电极/电解质的界面锂离子传输的改性方法。主要研究工作包括以下两个方面：
（1）在LiCoO2表面原位聚合一层聚α-氰基丙烯酸乙酯后，可以有效抑制LiCoO2/PEO/Li电池中PEO电解质在高工作电压时的分解反应，从而提高LiCoO2和PEO之间的界面电化学稳定性，改善电池的循环性能。
（2）在LiCoO2电极片中使用聚合物-无机复合电解质，有利于增加正极活性材料LiCoO2和PEO电解质之间的接触面积，降低电极和电解质之间的界面阻抗，提高界面锂离子传输能力，获得改进的循环稳定性。All-solid-state lithium ion battery (LIB) using high voltage cathode material is thought to improve the energy density and safety of recent commercial LIBs by replacing the liquid organic electrolyte with solid-state electrolyte. However, the interface impedance on the surface of electrode and electrolyte rises up in the all-solid-state LIBs, which will hinder the fast diffusion of lithium between electrode and electrolyte. Moreover, the high working voltage will challenge the interface stability between the electrode and electrolyte, and thus deteriorates the energy density and cycling stability of all-solid-state LIBs. Here, the interface issues and modification strategies in high voltage all-solid-state LIBs is investigated in the batter system of LiCoO2/polyethylene oxide (PEO)/Li.
Coating the LiCoO2 particles by in situ polymerization of ethyl a-cyanoacrylate improves the interface electrochemical stability between LiCoO2 and PEO by suppressing the decomposition of PEO electrolyte at high voltage and then enhances the cycling stability of LiCoO2/PEO/Li battery.
The polymer-inorganic composite electrolyte used in cathode electrode is benifical to decrease the interfacial impedance and improve the lithium ion diffusion between electrode and electrolyte. The electrochemical performance of LiCoO2/PEO/Li battery demonstrates enhanced cycling life
In Situ Formation of Polysulfonamide Supported Poly(ethylene glycol) Divinyl Ether Based Polymer Electrolyte toward Monolithic Sodium Ion Batteries
No full textThe effect of grain orientation on the morphological stability of the organic–inorganic perovskite films under elevated temperature.
No full text生物化工
No full textCaries is one of the most common infections in children and adult world-wide, however preventive intervention remains difficult. An accurate caries risk assessment method can identify patients at high caries risk for caries onset and thus help to deliver preventive therapies. Previous work in our research group showed that the spatial and temporal variation of oral microbiota can be employed for prediction of caries onset in children. However, the use of human cohorts have resulted in significant difficulties in mechanistic understanding of such predictive models, as in human cohorts it is difficult to precisely control the microenviroment and behavior of the individual subjects. The rodent model of dental caries, rats in particular, not only can simulate the onset and progression of caries in natural environments, but allows direct application and controls of the various etiological factors of caries, and thus greatly facilitate the longitudinal tracking of the microbiota dynamics during caries onset and progression. These advantages have made the rat model an excellent animal model for mechanistic study of the microbiota-based prediction of caries onset. However, previously there have been no reports that profile microbiome dynamics during caries development in rats.
Here we simultaneously tracked the longitudinal development of microbiota of 57 three-week-old rats for 12 weeks, during which 27 stayed healthy and 30 transited from health into cariogenesis. The techniques were mainly 16S rRNA gene amplicon-based pyrosequencing technology coupled with varies multivariate statistical analyses. Firstly, we found that the factors of age and status exert the key impact on the overall composition of the rat oral microbiota, and difference in the taxa level used had a strong influence on the analysis of variation of oral microbiota. Secondly, a host-aging correlated microbiota development pattern apparent in healthy stage was retarded by caries onset. Moreover, oral microbiota during caries onset was significantly more correlated with changes in disease severity than that during caries progression. Thirdly, by distinguishing between aging- and disease-associated OTUs and exploiting the distinct microbiota dynamics between the onset and progression phases of caries, Microbial indicators of Caries (MiC) based on Random Forests algorithm was proposed, which diagnosed caries from healthy samples with 99% accuracy, and furthermore predicted future new caries-onsets for those samples presently clinically perceived as healthy with 77% accuracy. Finally, we analyzed the link and distinction in microbiota development underlying cariogenesis between rat and human, and found that the temporal variation of the structure and microbial metabolism of the rat oral microbiota is consistent with human oral microbiota during caries and health, and the predictive model for caries onset based on Random Forests algorithm is robust in both human and rat oral microbiota. Interestingly, bacterial composition of oral microbiota at the genus level was different between human and rat, and the divergent evolution of human oral microbiota and rat oral microbiota was driven by status (caries) or age.
In summary, the findings in this M.S. thesis have several implications. Firstly, via the animal model of rat, this study further validates the notion that caries onset can be predicted via oral microbiota, and lays a theoretical foundation for future preventive intervention experiments based on such predictive model of diseases. Secondly, this study suggests that the microbiota data can be analyzed from microbiota structural changes and microbial metabolism, to analyze the experimental data from the microbiota of the animal models that are used to study how microbiota predicts disease. Finally, this study suggests that the Random Forests algorithm has huge potential for the analysis of the experimental data from the microbiota of the animal models and for the construction of disease predictive models.中
Efficient polymer solar cells based on a new quinoxaline derivative with fluorinated phenyl side chain
No full textA novel donor-pi-acceptor (D-pi-A) type polymer of PBDTT-DTFPQx composed of a medium electron-donating 5,8-dialkylthienyl substituted benzo[1,2-b:4,5-b']dithiophene (BDTT) moiety and a strong electron-accepting 4-fluorophenyl substituted 6,7-dioctyloxyquinoxaline (FPQx) with thiophene pi-bridge units was synthesized and characterized, as a donor material for polymer solar cells (PSCs). This polymer exhibits a low optical bandgap of 1.66 eV with an absorption onset of 745 nm, a low-lying HOMO energy level of -5.52 eV, and a hole mobility of 5.05 x 10(-4) cm(2) V-1 s(-1). Compared to the reported analogues, PSCs based on PBDTT-DTFPQx/PC71BM demonstrated an outstanding fill factor (FF) value. With an optimized blend ratio of PBDT-TFQ: PC71BM (1 : 4, w/w), a high power conversion efficiency (PCE) of 7.2% was obtained, with an open-circuit voltage (V-oc) of 0.87 V, a short-circuit current (J(SC)) of 11.4 mA cm(-2), and a FF of up to 73% under AM 1.5G irradiation. The results demonstrate that the introduction of two fluoride atoms onto 4-positions of the phenyl group at the quinoxaline unit by side-chain engineering into the BDTT-alt-DTQx type polymers would be a feasible approach to improve photovoltaic properties in PSCs
Gold nanoprobe functionalized with specific fusion protein selection from phage display and its application in rapid, selective and sensitive colorimetric biosensing of Staphylococcus aureus
No full textStaphylococcus aureus (S. aureus) is one of the most ubiquitous pathogens in public healthcare worldwide. It holds great insterest in establishing robust analytical method for S. aureus. Herein, we report a S. aureus-specific recognition element, isolated from phage monoclone GQTTLITS, which was selected from f8/8 landscape phage library against S. aureus in a high-throughput way. By fiinctionalizing cysteamine (CS)-stabilized gold nanoparticles (CS-AuNPs) with S. aureus-specific pVIII fusion protein (fusion-pVIII), a bifunctional nanoprobe (CS-AuNPs@fusion-pVIII) for S. aureus was developed. In this strategy, the CS-AuNPs@fusion-pVIII could be induced to aggregate quickly in the presence of target S. aureus, resulting in a rapid colorimetric response of gold nanoparticles. More importantly, the as-designed probe exhibited excellent selectivity over other bacteria. Thus, the CS-AuNPs@fusion-pVIII could be used as the indicator of target S. aureus. This assay can detect as low as 19 CFU mL(-1) S. aureus within 30 min. Further, this approach can be applicable to detect S. aureus in real water samples. Due to its sensitivity, specificity and rapidness, this proposed method is promising for on-site testing of S. aureus without using any costly instruments. (C) 2016 Elsevier B.V. All rights reserved