Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences
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Heterojunction-Depleted Lead-Free Perovskite Solar Cells with Coarse-Grained B-gamma-CsSnI3 Thin Films
No full textPerovskite solar cells (PSCs) have been emerging as a breakthrough photo-voltaic technology, holding unprecedented promise for low-cost, high-efficiency renewable electricity generation. However, potential toxicity associated with the state-of-the-art lead-containing PSCs has become a major concern. The past research in the development of lead-free PSCs has met with mixed success. Herein, the promise of coarse-grained B-gamma-CsSnI3 perovskite thin films as light absorber for efficient lead-free PSCs is demonstrated. Thermally-driven solid-state coarsening of B-gamma-CsSnI3 perovskite grains employed here is accompanied by an increase of tin-vacancy concentration in their crystal structure, as supported by first-principles calculations. The optimal device architecture for the efficient photovoltaic operation of these B-gamma-CsSnI3 thin films is identified through exploration of several device architectures. Via modulation of the B-gamma-CsSnI3 grain coarsening, together with the use of the optimal PSC architecture, planar heterojunction-depleted B-gamma-CsSnI3 PSCs with power conversion efficiency up to 3.31% are achieved without the use of any additives. The demonstrated strategies provide guidelines and prospects for developing future high-performance lead-free PVs
Methanol-to-olefin conversion over H-MCM-22 catalyst
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H-MCM-22 zeolites with different Si/Al ratios were hydrothermally synthesized, also the parent sample was dealuminated by ammonium hexafluorosilicate (AHFS). Then all of the samples were characterized and evaluated for the methanol-to-olefin (MTO) conversion. The correlation of structural and acidic properties of H-MCM-22 with catalytic performance was investigated. The H-MCM-22 (Si/Al= 35.1) exhibits best catalytic stability in all the samples because of its suitable acid site amount and distribution. As for the H-MCM-22 (Si/Al =35.1) after dealumination by AHFS, most of the extra-framework Al species were selectively removed, the selectivity of light olefin was enhanced further and the coke deposition seems to be inhibited. (C) 2015 Elsevier B.V. All rights reserved.</p
The thermophilic (55 degrees C) microaerobic pretreatment of corn straw for anaerobic digestion (vol 175, pg 203, 2015)
No full textPreparation, characterization and long-term antibacterial activity of Ag-poly(dopamine)-TiO2 nanotube composites
No full textA simple and efficient approach for the loading of Ag nanoparticles on poly(dopamine)-modified TiO2 nanotubes (PDA-TNTs) was used to prepare a Ag nanoparticle-poly(dopamine)-TiO2 nanotube composite (Ag-PDA-TNTs) that was applied as a long-term antibacterial agent to inhibit the growth of bacterial cells. The features of the obtained Ag-PDA-TNTs were investigated by TEM, XRD, FT-IR and XPS analysis. Anatase TNTs were encapsulated in PDA layers with a thickness of about 3 nm on which about 20.4 wt% of single crystalline Ag nanoparticles anchored. XPS and FT-IR results indicated that the PDA layer not only served as a reduction reagent that could reduce Ag+ ions to Ag nanoparticles but also worked as an adhesive coating by tethering the Ag nanoparticles on the surface of PDA-TNTs. The long-term release profiles of the nanocomposites showed that PDA layers slowed the release rate of Ag nanoparticles, implying the possible long-term antibacterial activity of Ag-PDA-TNTs; and the antibacterial assays verified this point. Besides that, the antibacterial activity of Ag-PDA-TNTs under visible light was higher than that in the dark because of the synergistically antibacterial effects of Ag nanoparticles and ROS under visible light irradiation. Compared with the antibacterial activity of TiO2 nanotubes loaded with Ag nanoparticles (Ag-TNTs), Ag-PDA-TNTs had higher and longer-term antibacterial activity which is attributed to the effect of PDA layers tethering Ag nanoparticles on TNTs and slowing Ag+ ions release rate. This work provides a new method for the preparation of Ag-based antibacterial agents and facilitates their practical application in modern antifouling and biomedical fields
Iridium nanoparticles supported on hierarchical porous N-doped carbon: an efficient water-tolerant catalyst for bio-alcohol condensation in water
No full textNitrogen-doped hierarchical porous carbons were synthesized successfully by a controllable one-pot method using glucose and dicyandiamide as carbon source and nitrogen source via hydrothermal carbonization process. The nitrogen-doped materials, possessing high nitrogen content (up to 7 wt%), large surface area (>320 m(2) g(-1)) and excellent hierarchical nanostructure, were employed as catalyst supports for immobilization of iridium nanoparticles for bio-alcohol condensation in water. The introduction of nitrogen atoms into the carbon framework significantly improved iridium nanoparticles dispersion and stabilization. The novel iridium catalysts exhibited superior catalytic activity in the aqueous phase condensation of butanol, offering high butanol conversion of 45% with impressive 2-ethylhexanol selectivity of 97%. The heterogeneous catalysts had great advantages of easy recovery and high catalytic stability. The outstanding catalytic performance could be attributed to excellent dispersion of iridium nanoparticles, stronger iridium-support interactions and interaction of nitrogen species with alcohol substrates
Enhanced efficiency of polymer solar cells by structure-differentiated silver nano-dopants in solution-processed tungsten oxide layer
No full textIn this article, we creatively incorporate naked Ag nanoparticles (nAgp), SiO2-covered Ag nanoparticles (SiAgp), and naked Ag nanoplates (nAgPl) into solution-processed tungsten oxide (WO3) hole transport layer in polymer solar cells (PSCs) to increase the light absorption through localized surface plasmon resonance (LSPR) effect. With optimized doping concentrations, nAgp decreases power conversion efficiency (PCE), however, SiAgp increases PCE by 13.2% and nAgPl increases PCE by 19.7%. In detail, the great PCEs discrepancies are mainly caused by the discrepancies of short circuit currents (J(sc)). The experiments results indicate that the naked surface of nAgp causes the exciton-quenching to decrease the J(sc) of PSCs. Whereas the capsulation of SiO2 at the surface of nAgp and the embedment of nAgPl in WO3 layer can prevent the exciton-quenching and help to use LSPR effect of Ag nano-dopants to enhance the J(sc) of PSCs. Crown Copyright (C) 2016 Published by Elsevier B.V. All rights reserved
High Conductive Two-Dimensional Covalent Organic Framework for Lithium Storage with Large Capacity
No full textA high conductive 2D COF polyporphyrin (TThPP) linked by 4-thiophenephenyl groups was synthesized through an in situ chemical oxidative polymerization on the surface of copper foil. The TThPP films were used as the anode of lithium-ion battery, which exhibited high specific capacities, excellent rate performances, and long cycle lives due to the alignment of 2D polyporphyrin nanosheets, and they (i) can highly efficiently adsorb Li atoms, (ii) have short-ended paths for the fast lithium ion diffusion, and (iii) open nanopores holding electrolyte. The reversible capacity is up to 666 mAh/g. This is the first example of an organic 2D COF for an anode of lithium-ion battery and represents an important step toward the use of COFs in the next-generation high-performance lithium ion battery
生物模板法可控合成过渡金属氧化物纳米结构及其电化学葡萄糖传感应用
No full text电化学葡萄糖传感器在血糖监测和工业生物过程监控中发挥着重要作用。电极材料及识别元件是电化学传感的基础，为了提高它们的性能，人们引入了纳米材料。其中，过渡金属氧化物由于其特殊的理化性能而被广泛应用于电化学葡萄糖传感。然而，传统的纳米材料合成方法通常存在着一些问题：产品结构简单、形貌单一；合成过程复杂、对操作人员要求高、耗能高、设备造价高、安全性差、原料成本高；所需的合成试剂或条件对环境或人体有害；电化学传感性能不理想。为克服这些挑战，本课题定位于利用绿色、简单、灵活、价廉的生物模板法合成具有高电化学传感性能的纳米材料以用于葡萄糖检测。
二氧化锰（MnO2）作为重要的过渡金属氧化物，具有独特的电催化及化学催化活性、绿色环保和无毒特性，已被广泛应用于电化学和分析化学领域。在MnO2的各种形貌中，一维的纳米形貌因其电子传递的限制结构、量子尺寸效应和表面效应，具有优秀的电化学应用潜力（比如超级电容器、离子电池和电化学传感器）。然而，它们的合成方法通常需要复杂的仪器设备、加热过程、有害的有机试剂和强氧化剂，从而限制了它们的应用。为了便捷地合成出形貌可控的一维MnO2纳米线，本课题以基因改造的丝状噬菌体M13作生物模板，通过Mn2+在碱性条件下的自发氧化，实现了MnO2晶体在噬菌体框架上的成核与生长。通过改变M13的表面电荷及浓度，可实现对纳米线形貌的控制，MnO2晶体能够均匀地分布在带负电荷的四聚谷氨酸融合噬菌体（M13-E4）上，而在野生型噬菌体和带正电荷的四聚精氨酸融合噬菌体上出现不规则团聚。所合成的M13-E4@MnO2纳米线在高盐中性溶液中展现了对H2O2的电催化氧化活性。为了进一步阐明此活性的优势，利用其和葡萄糖氧化酶构建了葡萄糖生物传感器。该传感器具有宽的线性范围（0.005~2 mM葡萄糖）、快速的响应（5 s之内）、可接受的检测限（1.8 μM，S/N = 3）、良好的批内批间再现性、满意的储存稳定性以及对实际样品检测的可靠性。由于其在合成及性能上的优越性，该MnO2纳米线有望应用于电催化剂、电化学传感、超级电容器等领域。
虽然上述生物传感器改善了常规酶传感器的一些性能，但它也存在一些缺点，如稳定性较差（主要归因于酶的使用）、灵敏度低等。因此，为了解决这些问题，我们选用了可作为葡萄糖电催化氧化剂的Co3O4来构建直接电化学无酶传感器。为了便捷地得到表面积大的直接电化学传感界面，本课题以银杏树叶为生物模板，开发了一种简单、价廉、环保的方法，从而合成了Co3O4三维多孔材料。该材料由相互交联的Co3O4颗粒（粒径30~100 nm）组成，从而产生了不规则的多孔结构，从而提供了更大的比表面积和电催化活性位点。随后，Co3O4三维多孔材料被成功地用作直接电化学传感界面以实现了对葡萄糖的无酶检测。所制备的传感器具有简单的制备步骤、快速的响应、较高的灵敏度（439.39 μA mM-1 cm-2）、较低的检测限（0.1 μM，S/N = 3）。另外，它也显示了优秀的稳定性、对血糖检测常见干扰物质（抗坏血酸、尿酸、多巴胺、对乙酰氨基酚）及氯离子的抗干扰性能、对血糖检测的可靠性。总之，由于其在合成及性能上的优越性，Co3O4三维多孔材料有望应用于电催化剂、电化学传感、超级电容器等领域。另外，所提出的材料合成方法具有普适性，鉴于不同的合成条件及不同的金属氧化物种类可能产生不同的纳米结构和特性，所开发的材料合成方法可以延伸出各种各样的金属或金属氧化物纳米材料并具有广阔的应用方向。Electrochemical glucose biosensor plays important roles in both diabete diagnostics and industrial bioprocess monitoring. As the foundation of the electrochemical sensor, the electrode materials or the recognition elements are improved by intruducing a variety of nanomaterials. Among the various nanomaterials, transition metal oxides have been widely applied on the electrochemical glucose sensor, due to their unique physicochemical properties. However, the conventional synthesis methods generally require complex process, expensive equipments and precursors, skilled workers, high consumption of energy, unsafe condition or harmful reagents. In addition, the nanomaterials from these methods lack complicated structure and morphology, and their electrochemical performances still need to be improved. To overcome these challenges, our aim is to prepare the novel nanomaterials with high electrochemical sensing performance by the green, simple, facile and cost-effective bio-templated strategy.
As a kind of attractive transition metal oxide, manganese dioxide (MnO2) with different morphologies, has been widely studied on the field of electrochemistry and analytical chemistry, due to its electrocatalytic activity, chemical catalysis, environmental friendliness and nontoxicity. Among the different morphologies of MnO2, one-dimensional (1D) nano-structured morphologies have attracted more and more interests for electrochemical applications (such as supercapacitor, ion battery and electrochemical sensor), due to the controlled structure for electron transfer, quantum size effect and surface effect. To conveniently obtain 1D MnO2 nanowires (NWs) with controlled structure and unique properties under mild conditions, the genetically engineered M13 phages were useed as templates for precise nucleation and growth of MnO2 crystals on filamentous phage scaffolds, via the spontaneous oxidation of Mn2+ in alkaline solution. It was found that the morphology of NWs could be tailored by the surface charge of M13 mutants. MnO2 crystals were uniformly distributed on the surface of negatively-charged tetraglutamate-fused phage (M13-E4), significantly different from irregular MnO2 agglomeration on the weakly negatively-charged wild-type phage and positively-charged tetraarginine-fused phage. The as-synthesized M13-E4@MnO2 NWs could catalyze the electro-oxidation of H2O2 at neutral pH. To demonstrate the superiority of the electrocatalytic activity in the solution containing plenty of chloride ions at neutral pH, both glucose oxidase and as-prepared MnO2 NWs were used for fabricating the glucose biosensor. The proposed biosensor showed a wide linear range (0.005~2 mM glucose), rapid response (within 5 s), an acceptable limit of detection (1.8 μM glucose, S/N = 3), good inter-and intra-assay reproducibility, satisfactory storage stability and reliability of real sample detection. Due to the superiorities on synthesis and electrochemical performance, the MnO2 NWs are promising to be applied on electrocatalysis, electrochemical sensor, and supercapacitor.
Although the above biosensor exhibits improved performance, there were still some disadvantages: low stability (mainly ascribed to enzymes) and low sensitivity. Therefore, we chose cobalt oxide (Co3O4), a kind of electrocatalyst of glucose oxidation, to fabricate the electrochemical non-enzymatic sensor. To obtain the direct electrochemical sensing interface with large surface area, a novel three-dimensional (3D) porous Co3O4 architecture was first synthesized through a simple, cost-effective and environmentally friendly leaf-templated strategy. The Co3O4 nanoparticles (30~100 nm) were interconnected with each other to form a 3D porous structure, which provided high specific surface area and numerous electrocatalytic active sites. Subsequently, Co3O4 was successfully utilized as direct electrochemical sensing interface for non-enzymatic detection of H2O2 and glucose. The fabricated Nafion/Co3O4/GCE glucose sensor showed not only the rapid response, high sensitivity (439.39 μA mM-1 cm-2) and low detection limit (0.1 μM glucose, S/N = 3), but also excellent stability, anti-interference performance for possible interferents (such as ascorbic acid, uric acid, dopamine, acetaminophen and especially 0.15 M chloride ions) and reliability for blood glucose detection. In conclusion, due to the superiorities on synthesis and performance, 3D porous Co3O4 is promising for possible applications in electrocatalysis, electrochemical sensor and supercapacitor. In addition, the proposed synthesis strategy may be applicable to prepare a wide range of metal or metal oxide nanostructures. Considering the different nanostructures and properties from different synthesis condition and metal oxides, various potential applications are envisioned
HZSM-5负载氧化锰催化剂的制备及其对VOCs催化氧化反应性能研究
No full text挥发性有机化合物（Volatile organic compounds，简称VOCs）是大气污染的主要来源之一，严重危害生态环境和人类健康。在诸多的VOCs处理技术中，催化氧化由于净化效率高、能耗低，且反应过程中将有机污染物直接转化为二氧化碳和水，被认为是最具应用前景的净化技术。目前，催化氧化技术的关键是获得具有优异催化性能且价格低廉的催化剂。尽管负载型贵金属催化剂表现出较高的低温催化活性，但是昂贵的价格限制了其实际应用。过渡金属氧化物对VOCs具有优良的高温催化活性和抗中毒性能，得到了研究人员的广泛关注。本论文以甲苯的催化氧化为模型反应，制备了一系列MnOx/HZSM-5催化剂并考察其催化氧化性能。采用X射线衍射（XRD）、扫描电子显微镜（SEM）、透射电子显微镜（TEM）、氮气物理吸附（N2 physisorption）、氨气程序升温脱附（NH3-TPD）和氢气程序升温还原（H2-TPR）等表征技术，研究了催化剂的物化性质和催化性能间的相互关系。论文主要取得了以下研究结论：
（1）采用等体积浸渍法，合成了具有不同负载量的y%MnOx/HZSM-5（y=2.5、5.0、7.5、10、15）催化剂。催化反应测试结果发现，随着MnOx负载量的增加，催化剂的催化活性逐渐提高；当负载量高于10%时，催化活性并没有得到提升反而下降。催化剂的催化活性由高到低依次为10%MnOx/HZSM-5>7.5%MnOx/HZSM-5≈15%MnOx/HZSM-5>5.0%MnOx/HZSM-5>2.5%MnOx/HZSM-5，其中10%MnOx/HZSM-5催化剂表现出了最优的催化活性（T50=261 ℃，T90=267 ℃）、良好的催化稳定性和抗水性能。HZSM-5分子筛表面的MnOx颗粒可以明显的抑制反应过程中积碳的生成并促进高温下积碳的分解，低温可还原性能是影响催化剂反应活性的关键因素。以商品级SiO2和Al2O3为载体制备的10%MnOx/SiO2和10%MnOx/Al2O3催化剂的催化活性均远低于10%MnOx/HZSM-5的催化活性。主要归因于HZSM-5分子筛中丰富的Brønsted酸中心，与MnOx颗粒氧化还原活性位间的协同作用显著地促进了甲苯催化氧化反应的进行。
（2）采用离子交换法合成了一系列具有不同Na/Al值的NaZSM-5分子筛，并以此为载体制备了相同负载量的10%MnOx/NaZSM-5-y（y=0.16、0.31、0.51）催化剂。各催化剂的总酸量和酸强度随着Na/Al值的增大而逐渐减小，而酸性的改变并未对其低温可还原性能造成明显的影响。在甲苯的催化氧化反应中，10%MnOx/HZSM-5催化剂仍表现出最好的催化活性和最低的表观活化能，且甲苯在单位锰活性位上的反应速率最高；然而随着催化剂酸量的减少，其对甲苯的催化活性也逐渐下降，单位锰活性位上的甲苯转化速率也逐渐降低；特别对于10%MnOx/NaZSM-5催化剂，其T50和T90相比10%MnOx/HZSM-5催化剂均高20 ℃以上。通过对物化表征结果与催化氧化活性综合分析发现，催化剂的催化活性随着总酸量的增加而提高，呈线性相关；催化剂较强的酸性有利于降低反应的表观活化能，对甲苯转化具有明显的促进作用。
（3）合成了具有不同硅铝比的HZSM-5分子筛，作为载体制备了相同负载量的10%MnOx/HZSM-5-y（y=25、50、75）。随着载体硅铝比的增加，催化剂的酸强度和总酸量逐渐降低。在甲苯的催化氧化反应中，10%MnOx/HZSM-5-25催化剂表现出最佳的催化活性和最低的表观活化能，其T50和T90分别为253 ℃和261 ℃。随着载体硅铝比的增加，其催化反应活性逐渐降低，再次证明了载体的酸性是影响催化剂整体催化性能的关键因素，更多的酸性位对甲苯的催化氧化反应具有显著的促进作用。
（4）合成了具有不同晶粒尺寸和内部结构的HZSM-5分子筛，考察了载体的尺寸效应对于MnOx负载型催化剂催化活性的影响。相比于微米尺寸的Micro-10%MnOx/HZSM-5催化剂，具有纳米结构的Nano-10%MnOx/HZSM-5催化剂具有更大的比表面积，有利于活性组分在其表面的分散，从而具有更好的可还原性能。对于Hollow-10%MnOx/HZSM-5催化剂，分子筛载体的中空结构能够进一步提高活性组分的分散性，使得催化剂的氢气还原温度大幅降低为277 ℃和375 ℃，表现出优异的氧化还原性能。甲苯催化氧化的反应结果发现，Hollow-10%MnOx/HZSM-5催化剂具有最佳的催化活性，其T50和T90分别为245 ℃和255 ℃，均远低于另外两个催化剂的甲苯转化温度。其最低的表观活化能（Ea=101.3 kJ mol-1）表明在较低的反应温度下更易于发生甲苯的转化。中空结构的存在，使催化剂的外表面积和介孔体积都有显著的增加，这不仅有利于活性组分MnOx颗粒在载体表面的分散，提高催化剂的还原性能并进一步提高其甲苯催化氧化活性，而且还缩短了反应过程中各物质的传递距离，促进了催化氧化反应的进行。Volatile Organic Compounds (VOCs), one of the main sources of air pollution, pollute the environment and are harmful to human health. Various applicable technologies have been developed in order to efficiently remove VOCs pollutants. Among all, catalytic oxidation has been proven to be an effective and energy-saving process where organic pollutants could be completely decomposed into those harmless products such as carbon dioxide and water at relatively low temperature. The main concern in the field of VOCs catalytic oxidation is to search a proper catalyst with high catalytic activity and stability but low cost. In spite that supported noble metals are highly active in catalyzing VOCs oxidation at low temperature, high costs and easy deactivation are the main defects limiting their wide application. It has been accepted that transition metal oxides are another branch of catalysts for VOCs oxidation because of their good catalytic activities at high temperature and resistant ability to poisoning. In this thesis, catalytic performances of MnOx/HZSM-5 catalysts were evaluated for the catalytic oxidation of toluene, which was typically selected as a model reaction for ordinary VOCs abatement. Numerous characterization techniques including XRD, N2 physisorption, SEM, TEM, H2-TPR and NH3-TPD were conducted to investigate the correlation between the physicochemical properties and the catalytic performances over the prepared catalysts. The main results obtained in the thesis are as follows:
(1) Series of y%MnOx/HZSM-5 (y=2.5, 5.0, 7.5, 10, 15) catalysts were successfully prepared by an incipient impregnation method and used for the catalytic oxidation of toluene. Catalytic testing results indicated that the catalytic activities of HZSM-5 supported MnOx catalysts decreased in the order of 10%MnOx/HZSM-5> 7.5%MnOx/HZSM-5≈15%MnOx/HZSM-5> 5.0%MnOx/HZSM-5> 2.5%MnOx/ HZSM-5, where 10%MnOx/HZSM-5 exhibited the optimum catalytic activity, excellent catalytic durability in dry condition as well as good regeneration capability in humid condition. The dispersion of MnOx species on HZSM-5 surface greatly reduced the formation of coke and simultaneously efficiently accelerated the oxidative elimination of surface coke deposition during the catalytic oxidation of toluene which was the factor for toluene oxidation reaction. Based on a comparative analysis with those SiO2 and Al2O3 supported MnOx catalysts, it was predicated that the Brønsted acid sites of HZSM-5 zeolite make a promotional effect on the catalytic oxidation of toluene. The cooperative action between the redox ability of MnOx species and the acidity property of HZSM-5 zeolite resulted in high catalytic reactivity for toluene oxidation.
(2) Using the ion exchange method, series of 10%MnOx/NaZSM-5-y (y=0.16, 0.31, 0.51) catalysts were prepared. The total acidity of the catalysts was decreased with the increasing of Na/Al ratio and there was no obvious relationship between the acdity and the reductibility for the catalysts. By comparing to the ion changed samples, 10%MnOx/HZSM-5 catalyst showed the best catalytic performance with the lowest Ea value. After a comparative analysis and discussion based on the obtained characterizations and catalytic testing results, it showed that the acidity of catalysts highly improved the catalytic activities in the toluene oxidation reactions by lowering the activation energy.
(3) A range of HZSM-5 zeolite with various Si/Al ratio (25, 50, 75) suppored MnOx catalysts had been successfully prepared via hydrothermal systhesis and impregnation. The Si/Al ratios strongly influenced the acidity of 10%MnOx/ HZSM-5-n (n=25, 50, 75) catalysts. Catalytic tests in toluene oxidation showed that 10%MnOx/HZSM-5-25 exhibits the highest activity (T50=253 ℃ and T90=261 ℃) and the lowest activation energy value among all the catalysts. The catalytic activity was reduced with the increasing of Si/Al ratio, indicating that actidiy of the catalyst is the key factor responsible for the catalytic activity.
(4) In order to investigate the influence of size effect on the catalytic activites, series of catalysts with different partical sizes and inner structure were prepared. Campared with micro-sized sample Micro-10%MnOx/HZSM-5, the Nano-10%MnOx/ HZSM-5 catalyst exhibited a better reducing property which was attribute to its higher specific surface area. In the catalytic tests for toluene oxidation, Hollow-10%MnOx/ HZSM-5 catalysts exhibited the best catalytic activity (T50=253 ℃ and T90=261 ℃) and the lowest activation energy value. The special hollow structure significant improved the dispersion of MnOx on the catalyst surface, lowered the reaction activation energy and promoted the toluene oxidation. In addition, the short transfer route was another advantage of the hollow structure
大肠杆菌中3-羟基丙酸抗性机制及其衍生物丙烯酸的生物合成研究
No full text3-羟基丙酸（3-HP）是一种重要的平台化合物，拥有成熟的生物合成方法，但是其工业化应用方面存在两个主要问题：1，3-HP对细胞有毒性抑制，影响细胞生长，增加生产成本；2，部分3-HP衍生物人工代谢途径缺失。本文针对这两个问题，研究大肠杆菌对3-HP的应激过程，筛选到一批应激基因，发现一种新的酸抗性机制，并通过代谢工程改造大肠杆菌合成了3-HP的重要衍生物—丙烯酸。
为了解大肠杆菌的3-HP抗性机制，该研究在3 g/L 亚致死浓度3-HP存在时，利用单基因缺失文库keio collection （NBRP，日本）监测每个突变株在3-HP胁迫时的生长状况。使用基本培养基培养并测定突变株文库的生长曲线，结果显示3-HP胁迫时，有18个单基因缺失株耐受性增强，有20个单基因缺失菌株对3-HP更敏感。在此基础上将筛选得到的菌株在对数中期OD600nm~0.6时加入5 g/L 3-HP胁迫，测定以上菌株在对数中期是否有同样的效应。存活率结果显示，4个单基因缺失菌株依然表现出耐受性增强的性状，7个单基因缺失菌株表现为对酸很敏感。
本研究发现3-HP刺激后，大肠杆菌细胞膜磷脂中不饱和脂肪酸组分增多，利用其他有机酸和无机酸刺激后确认这种变化是由3-HP的酸毒性所导致，根据此结果本研究对酸刺激与不饱和脂肪酸合成之间的关系进行研究。存活率测定显示，酸刺激时不饱和脂肪酸合成关键基因fabA、fabB对细胞的酸耐受性至关重要，通过生物学信息预测，在fabA、fabB的启动子区发现了CpxR保守结合位点的类似序列。通过对fabA和fabB在转录和蛋白水平表达量的分析，结合存活率实验，最终证明二组分系统CpxRA可以上调fabA、fabB的转录和蛋白表达水平, 进而导致大肠杆菌细胞膜磷脂中不饱和脂肪酸含量升高，增强细胞对酸胁迫的耐受性。实验结果表明我们在大肠杆菌中发现了一种新的酸抗性机制，本发现将会进一步完善大肠杆菌酸抗性体系，对有机酸的生物合成及部分细菌的病理学研究有重要意义。
在3-HP的衍生物中，丙烯酸作为重要的有机合成原料及合成单体，其人工合成路径缺失。从分子结构来看，3-HP与乳酸是同分异构体，都可以直接脱水形成丙烯酸，但是由于C3结构稳定性强，不易直接在碳链骨架上进行氧化还原等反应，故而需要在其末端加上CoA基团，活化C3结构，使相关反应更易进行。该研究首次设计了一条依赖辅酶A的新路径，以甘油为底物和碳源，经由3-羟基丙醛，3-羟基丙酰辅酶A，丙烯酰辅酶A，最终在大肠杆菌中直接发酵得到丙烯酸。新路径的限速酶是3-羟基丙酰辅酶A脱水酶，其属于烯酰辅酶A水合酶家族，通过序列比对及文献调研，共找到4个候选的烯酰辅酶A水合酶：PhaJ1, EcH, HcaD, PcsII。将这4个烯酰辅酶A水合酶分别引入新的代谢路径，检测更适合本途径丙烯酸合成的酶，结果发现PcsII更适合在本研究中作为3-羟基丙酰辅酶A脱水酶生产丙烯酸。引入PcsII之后，新的路径能够代谢产生丙烯酸，但发酵产量很低，为了提高产物量，本研究将双质粒中的部分基因整合到工程菌株染色体上，携带单质粒的工程菌摇瓶发酵得到6.92 mg/L产物。本研究还对PcsII进行改造以提升丙烯酸产量，通过与烯酰辅酶A水合酶家族有报道的蛋白质结构信息比对，发现两个对于烯酰辅酶A水合酶家族保守的氨基酸残基：Lys和Ala，利用定点突变技术将PcsII 103位的Arg和186位的Asp分别回复突变为Lys和 Ala。突变后发现产物量有较大提升，将两个突变位点整合在PcsII上形成双突变PcsIIR103K-D186A，最终使摇瓶中丙烯酸的产物量达到37.7 mg/L。3-Hydroxypropionate (3-HP) is an important platform chemical, and its biosynthesis method is mature, however，there are two major problems block industrial application in biosynthesis: 1, 3-HP toxicity suppress the growth of cells, affect cell growth, increase total production cost; 2, some 3-HP derivatives lack artificial metabolic pathways. In order to solve the two problems, the study has focus on the stress process of E.coli to 3-HP. As a result, we got a cluster genes response to 3-HP, and discovered a new acid resistant mechanism in E.coli, at the same time, we synthesized an important 3-HP derivative-acrylic acid by using metabolic engineering in E.coli.
Organic acids have toxicity repress on the growth of bacteria, in order to understand the mechanism of 3-HP resistance in E.coli, this study employed single gene mutant library (keio collection, NBRP, Japan) to monitor the influence of each mutant. when cells after adaption to 3 g/L 3-HP (pH 4), the growth curve of E.coli were tested in M9 medium, The results showed that 18 mutants become more tolerance and 20 mutants had opposite effect when cells were adapted to 3-HP. To test if same effect of above strains in the middle of the logarithmic phase, the screening strains were conducted acid shock (5 g/L 3 - HP) when cells were in the middle of the logarithmic phase( OD600nm ~ 0.6 ). The result of percentage of survival revealed, 4 mutants still show more tolerance and 7 mutants were sensitive when expose 3-HP.
The study revealed that two-component system CpxRA elements cpxA and cpxR mutant was more sensitive when expose to acids shock, separately. Meanwhile, after adaption to pH, a change in the membrane lipid composition of E.coli was observed. We analyzed the promotor region of fabA and fabB, and a similar conserved sequence of CpxR recongnition site was found. fabA and fabB were critical enzymes of unsaturated fatty acids in E.coli. In the study, we demonstrated that the two-component system CpxRA is directly activated by acid shock, and the CpxRA system could up-regulates transcription level of fabA and fabB, and the UFAs biosynthesis was up-regulated in E.coli under acidic conditions. In conclusion, a new acid resistance system was found in E.coli, and the new acid resistance will help for prevention and clinical treatment and promoter the yield of organic acid in biosynthesis.
Acrylic acid is an important target for fermentative production, as an important organic synthetic raw materials and synthetic resin monome. However, there is not artificial production pathway to produce acrylic acid in engineering strain. 3-HP and lactic acid are isomers, in theory, it maybe generate acrylic acid from 3-HP or lactic acid by dehydrogenation. But in fact, the plan proves infeasible because the C3 structure is very stable, it is hard to conduct redox reaction in the carbon chain skeleton. It’s need to ligate a CoA group at the end of C3 skeleton to activate lactic acid structure, only by this way can dehydrogenation reactions are more likely to perform. For the first time, the study designed a new pathway, and glycerol as substrate, through 3-hydroxypropylaldehyde, 3-hydroxypropionyl coenzyme A, acryloyl coenzyme A, at the end acrylate was produced by E.coli fermentation. 3-hydroxypropionyl CoA dehydrogenase is rate-limiting enzyme in the new pathway, so it is necessary to screen efficient 3-hydroxypropionyl CoA dehydrogenase. According to the reactions, 3-hydroxypropionyl CoA dehydrogenase belongs to enoyl coenzyme A hydratase superfamily. In this study, four candidates of enoyl coenzyme A hydratase were picked out via sequence alignment and literature investigation, and there name were: PhaJ1, EcH, HcaD, PcsII. In order to analyze whcih enoyl-CoA hydratase was more appropriate for the production of acrylic acid, Four candidate enzymes were introduced into new metabolic pathways respectively. The results revealed that PcsII is more suitable as 3-hydroxypropionyl CoA dehydrogenase in the pathway. Although acrylate could be detected after PcsII was introdued in the new pathway, the production of shake flask fermentation yield were quited low. In order to improve the product, chromosomal gene integration (CGI) was employed which two genes were integrated into chromosome in engineering strain, and 6.92 mg/L acrylate were detected by shake flask. After CGI was employed, the product was still low, we speculate the production were repressed by low catalytic efficiency of PcsII. Alignment of PcsII with known structure of the enoyl-CoA hydratases，two amino acid residues were found conserved in all other proteins but not in PcsII：Lys and Ala. So, site-directed mutagenesis was performed to construct PcsII single mutants, and the effect of point mutations was verified by shake flask fermentation. Strains with PcsIIR103K and PcsIID186A produced 16.1 mg/L and 20.8 mg/L acrylic acid, respectively. When the double point mutations R103K/D186A were constructed in PcsII, the titer of acrylic acid was increased to 37.7 mg/L