在这个视频文章进行的实验，开发了国际基因机器设计竞赛9。想感谢iGEM比赛的团队成员，卢克Bergwerff，彼得TM面包车Boheemen，Jelmer克诺森，乌戈·F.奎托·罗哈斯和拉蒙·Van Der Valk酒店协助这项研究。我们感谢韩Winde，斯特凡去角和Esengül的的耶尔德勒姆的有益讨论和主持这项研究。这项工作是支持的代尔夫特理工大学生物技术系，生物信息学实验室的代尔夫特，代尔夫特理工大学的Bionanoscience，油砂领导倡议（OSLI），梭哈studentenuitzendbureau，荷兰基因组计划，Kluyver中心，荷兰Biotechnologische Vereniging（STICHTING生物技术荷兰） ，DSM，香港传艺，格雷纳生物和杰能科。

1。 BioBrick大会<ol><li> BioBricks标准生物部件登记处提供的iGEM比赛的总部。构造一个新的BioBrick从现有BioBricks，与酶EcoRI和SpeI消化的的捐助BioBrick（高达1.0微克），用于定位的给体部分的受体部分的下游。用XbaⅠ和PstⅠ月刊用于定位给体部分上游的受体部分。添加第三个适当的限制性内切酶切割的捐赠者中的骨干。在用适当的缓冲液的总体积为20-25毫升，根据供应商（最终浓度:1倍）下进行消化。使用5个单位/微克DNA的限制性内切酶。 </li><li>消化受体BioBrick与EcoRI和XbaI或SpeⅠ和PstⅠ位。 </li><li>孵育消化（至少）一个小时，在37°C。通过热灭活的限制性内切核酸酶，温育在80℃下10分钟，并离心不久。 </li><li>结扎消化BioBrick部分（供体和受体）。由于XbaI和SpeI位产生兼容的DNA末端，创建混合站点不能被任何限制性内切酶切割产生一个新的"组合拳"BioBrick，两侧的4个标准的限制性酶切位点。在连接混合物的最终DNA浓度优选为〜100纳克/微升。执行用T4连接缓冲液（最终浓度:1倍）和T4连接酶（1单位/微克DNA）的总体积为10-15毫升的连接反应。 </li><li>连接混合物在16℃孵育至少3小时。 </li><li>大约一半的连接混合物进行转化反应。 </li><li>确认BioBrick与测序是正确的。 </li><li>要建立一个新的BioBrick合成的DNA，修改合成的基因在大肠杆菌中的优化表达大肠杆菌通过工具（ <a href="http://www.jcat.de/" target="_blank">http://www.jcat.de/</a> ）的JCAT网站。按照要求的B中，进行进一步的修改<html> ioBrick标准。这种标准化意味着每个BioBrick是之前由一个前缀和后跟一个后缀的利息的DNA序列组成。前缀和后缀的序列包含预定义的酶切位点，在剩下的质粒序列所没有的。这些标准的限制性位点，使人们有可能互换和弹性延长BioBrick插入9。前缀和后缀有下列序列: </li></ol><table border="1"><tbody><tr><td> 名称 </td><td> 序列 </td><td> 评论 </td></tr><tr><td>字首</td><td> 5&#39;GAATTCGCGGCCGCTTCTAG3" </td><td></td></tr><tr><td></td><td> 5&#39;GAATTCGCGGCCGCTTCTAGAG 3&#39; </td><td>如果下面的部分是一个编码序列或任何部分开始"ATG" </td></tr><tr><td>后缀</td><td> 5&#39;TACTAGTAGCGGCCGCTGCAG 3&#39; </td><td></td></tr></tbody></table>ove_title"&gt; 2。烷烃转化静息细胞含量， 在体内 Fujii 等人 （2004）所记载的方法的基础上，进行该测定。 <ol><li>文化E.大肠杆菌表达烷烃羟化酶的系统（ <a href="http://partsregistry.org/Part:BBa_K398014" target="_blank">BBa_K398014</a> ）和细胞携带一个空的的向量（ <a href="http://partsregistry.org/Part:BBa_J13002" target="_blank">BBa_J13002</a> ）的5毫升LB培养基中，与适当的抗生素过夜。 </li><li>转移到50毫升的新鲜LB（与抗生素）和500微升过夜培养孵育直到细胞浊度达到在600nm处的OD（光密度）为0.3。 </li><li>在4,000 rpm下离心10分钟，将沉淀重悬于5毫升0.1M的磷酸盐缓冲液（pH7.4）。 </li><li>于4,000 rpm离心10分钟，再次离心，将沉淀重悬于在5ml 0.1M磷酸缓冲液现在包含E2的盐和0.66％v / v甘油（氮缺陷T培养基）。 </li><li>测细胞的浊度（OD 600）。 </li><li>准备6毫升的细胞混合物的等分试样在25毫升封闭帽的玻璃烧瓶中并没有细胞对照组（E2盐+ 0.66％v / v甘油）。 </li><li>向每个烧瓶中加入100 nmol的烷烃。 </li><li>孵育24小时缩短率较高时，获得的混合物在37°C。 </li><li>测量外径600后孵化。 </li><li>提取物在培养基中的烃由乙酸乙酯中，用气相色谱法（见协议3），并确定在细胞培养中的烃浓度。 </li><li>除以转换每个烧瓶中（OD 600转换到干重）的总生物量中存在的链烷烃的总量，计算每单位的生物量的退化。实验的持续时间将结果除以每单位生物量，每单位时间产生的降解活性。平均取自三个单独的运行。 </li></ol> 3。烷烃转化酶活性检测， 在体外基本上按照由Li 等人 （2008年）记载的方法进行该测定。 <ol><li>文化E.大肠杆菌细胞表达:LADA基因（ <a href="http://partsregistry.org/Part:BBa_K398017" target="_blank">BBa_K398017</a> ）和细胞携带一个空的的向量（ <a href="http://partsregistry.org/Part:BBa_J13002" target="_blank">BBa_J13002</a> ）的在50毫升LB培养基中过夜与适当的抗生素。 </li><li> 500微升过夜培养转移到50毫升的新鲜LB（与抗生素），并孵育，直到细胞浊度达到在600nm处的光密度为0.6。 </li><li>在4,000 rpm下（4℃）离心10分钟，将沉淀重悬于5毫升的50mM Tris缓冲液中。 </li><li>超声处理的细胞（细胞破碎，LA生物系统公司）在40％占空比的输出控制4，保持解决方案的整个持续时间上冰。 </li><li>离心所得到的混合物于4,000 rpm离心5分钟，在4及d例如，C，以除去细胞碎片。将上清液转移到一个新的小瓶。 </li><li>确定通过Bradford测定法的细胞提取物中的总蛋白浓度。注意:使用玻璃小瓶中，以防止增加的背景和/或损失的蛋白质。 </li><li>准备的100毫升的混合物，含有0.1％（体积/体积）的烷烃和50mM Tris-HCl缓冲。 </li><li>加热该混合物，在100℃下5分钟。注意:执行此步骤仅适用于中高沸点的长链烷烃。 （ 例如:C 16:287°C）。 </li><li>为了达到最佳的烷烃中的溶解度，超声处理1分钟，同时仍然温暖，直到得到均匀的，粘稠的混合物。 </li><li>加入1mM的FMN的NADH，1mM的，1 mM的MgSO 4和0.01体积/体积的Triton X-100。 </li><li>准备6毫升等分封闭帽的在25毫升烧瓶。 </li><li>添加足够量的细胞提取物（取决于Bradford分析，最终浓度为5毫克蛋白/升）。准备一个没有控制蛋白质。 </li><li>孵育在60°C（最佳Ënzymatic活动）为24小时。 </li><li>提取物在培养基中的烃由乙酸乙酯中，用气相色谱法（见协议3），并确定在细胞培养中的烃浓度。 </li><li>计算每单位的生物量除以烷烃的总量由总蛋白质转换退化加入到每个烧瓶中（通过Bradford校准曲线）。进一步除以实验的持续时间，产生每单位每单位时间的细胞蛋白的降解活性。平均取自三个单独的运行。 </li></ol> 4。醋酸乙酯油气开采和浓度测量<ol><li>从水溶液中萃取链烷烃的加入2.5毫升乙酸乙酯（非极性solvant）到6毫升实验溶液。内部标准被添加到溶剂中，在浓度为0.1％（体积/体积）。的标准（ 例如，环癸烷）的变化取决于景点的峰的预期范围。 </li><li>可选:添加的TritonX-100的含水混合物，并在4,000 rpm下的10分钟离心处理，以使样品，以获得适当的双相系统。 </li><li>振荡，持续5秒（1,500 rpm下）和在室温下孵育，直到混合物的两相分开。 </li><li>删除最大量的有机层（顶部）和干燥的溶剂中，用无水硫酸镁干燥。 </li><li>删除用MgSO 4通过过滤（0.2微米），并转移到气相色谱仪小瓶滤液进行测量，或存储于-20℃下</li><li>确定的浓度用气相色谱法，使用CP-SIL 5CB柱（长度= 5米）。以分流模式注入10μl的样品（1:10，230°C）。设置列的气体流量，以1毫升/分钟（氦）。下面的烘箱温度程序是用来: <table border="1"><tbody><tr><td> 率 </td><td> 温度[°C] </td><td> 时间[min] </td></tr><tr><td> 0 </td><td> 50 </td><td> 7.5 </td></tr><tr><td> 50 </td><td> 90 </td><td> 1.0 </td></tr><tr><td> 50 </td><td> 110 </td><td> 2.0 </td></tr><tr><td> 50 </td><td> 130 </td><td> 2.0 </td></tr><tr><td> 50 </td><td> 145 </td><td> 2.0 </td></tr><tr><td> 50 </td><td> 160 </td><td> 2.0 </td></tr><tr><td> 50 </td><td> 170 </td><td> 2.0 </td></tr><tr><td> 50 </td><td> 185 </td><td> 2.0 </td></tr><tr><td> 50 </td><td> 210 </td><td> 2.0 </td></tr><tr><td> 50 </td><td> 250 </td><td> 2.0 </td></tr><tr><td> 50 </td><td> 320 </td><td> 2.0 </td></tr></tbody></table></li><li>峰进行积分并纠正的浓度相对于内部标准。 </li></ol> 5。醇/醛脱氢酶活性测定基本上按照由Kato 等人描述的方法进行该测定。（2010年）。 <ol><li>文化E.大肠杆菌细胞表达的的ADH（ <a href="http://partsregistry.org/Part:BBa_K398018" target="_blank">BBa_K398018</a> ）或乙醛脱氢酶 （ <a href="http://partsregistry.org/Part:BBa_K398030" target="_blank">BBa_K398030</a> ）基因和细胞携带一个空的的向量（ <a href="http://partsregistry.org/Part:BBa_J13002" target="_blank">BBa_J13002</a> ）的在50毫升LB培养基中，与适当的抗生素过夜。 </li><li> 500微升过夜培养转移到50毫升的新鲜LB（与抗生素），并孵育，直到细胞浊度达到在600nm处的光密度为0.6。 </li><li>在4℃下以4,000 rpm离心10分钟，将沉淀重悬于50mM Tris缓冲液的5毫升。 </li><li>与4的输出控制（保持该溶液在冰上超声处理过程中），超声处理的细胞溶液在40％的占空比。 </li><li>在4,000 rpm下离心5分钟，将所得混合物在4℃下以除去细胞碎片。 </li><li>确定的TAL蛋白的细胞提取物的浓度由Bradford法（注:使用的玻璃小瓶，以防止蛋白结合）。 </li><li> 96孔板中，用1​​80微升57 mM甘氨酸缓冲液含有NAD（终浓度1mM，pH 9.5）中的各孔中装入。 </li><li>加入5μl的醇（醛）进行测试，以井。注:热的长链醇的测定开始前，有一个液体的。对于每个醇（醛）基板的控制而不应该被添加（阴性对照）。此外，含有无细胞提取物的混合物的缓冲液和基板制备空白。 </li><li>预热板读板器（Tecan公司麦哲伦v7.0的）为15分钟，在37℃至允许系统的平衡。 </li><li>添加足够量的细胞提取物（取决于Bradford分析，最终浓度为5毫克蛋白/升）。准备一个没有控制蛋白质。 </li><li>测量的NADH的生产用分光光度计在波长340nm的每2-3分钟1小时，在37℃下&lt;/ P&gt; <li>从OD（340 nm）的斜率计算的NADH的生产速率。考虑到光路长度和6220 M -1 cm -1的消光系数的NADH。的NADH的生产速率除以蛋白质的总量添加到表达脱氢酶反应的细胞提取物中的活性（U /毫克全细胞蛋白）。计算从三个独立运行的平均值和标准偏差。 </li></ol> 6。 pCaiF表征<ol><li>文化E.大肠杆菌细胞表达pCaiF-GFP的结构（ <a href="http://partsregistry.org/Part:BBa_K398331" target="_blank">BBa_K398331</a> ）和细胞的启动子（ <a href="http://partsregistry.org/Part:BBa_K398326" target="_blank">BBa_K398326</a> ）在5毫升LB培养基过夜适当的抗生素隔夜。 </li><li>接种用50μl的过夜培养物中含有10克/升葡萄糖和抗生素5毫升M9，和成长过夜。 </li&gt; <li>继代培养的50微升的通宵生长细胞浊度达到到5毫升新鲜的M9 with10克/升并孵育，直到在600nm处的光密度为0.2。 </li><li>装入的96孔板用100μl的新鲜的M9培养基中，含有所需量的碳源，在各孔中进行测试。执行一式三份实验进行统计评价，添加相应的阴性对照（野生型大肠杆菌 K12）。 </li><li>添加5μL过夜培养介质中井。 </li><li>测量的生长曲线（OD 600）和GFP荧光（485 nm激发和520排放）使用读板器每10分钟18小时，在37℃下恒定摇动。 </li><li>计算的增长速度，从各自的测量的特定GFP内容的控制比较。计算至少三个独立实验的平均值和标准偏差。 </li></ol> 7。公差含量<ol><li>文化大肠杆菌表达PhPFDα和β基因（ <a href="http://partsregistry.org/Part:BBa_K398406" target="_blank">BBa_K398406</a> ）的隔夜在5毫升LB培养基和适当的抗生素。E.大肠杆菌表达: 拉达基因（ <a href="http://partsregistry.org/Part:BBa_K398017" target="_blank">BBa_K398017</a> ）作为阴性对照。 </li><li>继代培养的10μl的产物到新鲜的5毫升LB（与抗生素）并孵育过夜，直到细胞浊度达到的光学密度为0.3至0.4，在600 nm。 </li><li>用新鲜培养基稀释培养，直到OD 600达到0.1。 </li><li>装入用180微升含有适当的抗生素和适当的最终浓度的有毒化合物（ 例如，0，4，8，10％的 n-己烷），一式三份的M9培养基中，在96孔板上。由于烷烃-水混合溶剂可能会导致两相系统，它是必不可少的，盘上的适当的控制（ 如d，增添菌株和各自的空白实验）。 </li><li>加入20μl的培养物（对照）入井。 </li><li>测量生物量浓度（OD 600）使用读板器每10分钟24小时在37℃下以恒定摇动。 </li><li>从各自的不同剂浓度的测量计算的生长率和阴性对照进行比较。计算的平均值和标准偏差至少三名独立运行的。 </li></ol> 8。同源互动映射<ol><li> STRING数据库（免费供教学使用）20上运行PostgreSQL服务器的应用他进行蛋白查询。的同源性互动地图应用程序标识使用STRING数据库在原有的宿主生物体相互作用的蛋白。用于在BLAST搜索找到同源基因中的靶生物的各相互作用的基因的序列。 Cytoscape的4 </s向上&gt;是用于可视化的结果的映射。他的软件工具可以下载: <a href="https://github.com/jcnossen/InteractionHomologMapping" target="_blank">https://github.com/jcnossen/InteractionHomologMapping。</a> </li><li>要进行映射，（1）输入BioBrick的ID和应用程序将自动下载标准生物零件，或（2）输入（粘贴）序列数据在应用程序从注册表中的部分序列数据。 </li><li>使用STRING数据库网站输入的氨基酸序列的找到STRING蛋白质的ID。 </li><li>使用BLAST确定一种蛋白质，具有较高的同源性。随后，应用程序列出了每个已知的相互作用蛋白在宿主生物中的同源物（ 如大肠杆菌 ）在源生物和搜索。 </li><li>假定的互动列表导出到文本或Cytoscape的。 </li></ol>

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	<li>Allen, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Process+water+treatment+in+Canada's+oil+sands+industry:+I:+Target+pollutants+and+treatment+objectives.">Process water treatment in Canada's oil sands industry: I: Target pollutants and treatment objectives.</a> J. Environ. Eng. Sci. 7, 123-138 (2008).</li><li>Alon, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> An Introduction to Systems Biology: Design Principles of Biological Circuits. , (2007).</li><li>Center, O. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Understanding Oil Spills and Oil Spill Response. , (1999).</li><li>Eichler, K., Buchet, A., Lemke, R., Kleber, H. P., Mandrand-Berthelot, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+the+caiF+gene+encoding+a+potential+transcriptional+activator+of+carnitine+metabolism+in+Escherichia+coli.">Identification and characterization of the caiF gene encoding a potential transcriptional activator of carnitine metabolism in Escherichia coli.</a> J. Bacteriol. 178, 1248-1257 (1995).</li><li>Feng, L., Wang, W., Cheng, J., Ren, Y., Zhao, G., Gao, C., Tang, Y., Liu, X., Han, W., Peng, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+and+proteome+of+long-chain+alkane+degrading+Geobacillus+thermodenitrificans+NG80-2+isolated+from+a+deep-subsurface+oil+reservoir.">Genome and proteome of long-chain alkane degrading Geobacillus thermodenitrificans NG80-2 isolated from a deep-subsurface oil reservoir.</a> Proc. Natl. Acad. Sci. U.S.A. 104 (13), 5602-5607 (2007).</li><li>Fujii, T., Narikawa, T., Takeda, K., Kato, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biotransformation+of+various+alkanes+using+the+Escherichia+coli+expressing+an+alkane+hydroxylase+system+from+Gordonia+sp.+TF6.">Biotransformation of various alkanes using the Escherichia coli expressing an alkane hydroxylase system from Gordonia sp. TF6.</a> Biosci. Biotechnol. Biochem. 68 (10), 2171-2177 (2004).</li><li>Kato, T., Miyanaga, A., Haruki, M., Imanaka, T., Morikawa, M., Gene Kanaya, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=cloning+of+an+alcohol+dehydrogenase+from+thermophilic+alkane-degrading+Bacillus+thermoleovorans+B23.">cloning of an alcohol dehydrogenase from thermophilic alkane-degrading Bacillus thermoleovorans B23.</a> J. Biosci. Bioeng. 91 (1), 100-102 (2001).</li><li>Kato, T., Miyanaga, A., Kanaya, S., Morikawa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+cloning+and+characterization+of+an+aldehyde+dehydrogenase+from+long-chain+alkane-degrading+Geobacillus+thermoleovorans+B23.">Gene cloning and characterization of an aldehyde dehydrogenase from long-chain alkane-degrading Geobacillus thermoleovorans B23.</a> Extremophiles. 14, 33-39 (2010).</li><li>Kieboom, J., De Bont, J. a. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Bacterial Stress Responses. , (2000).</li><li>Kotte, O., Zaugg, J. B., Heinemann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+adaptation+through+distributed+sensing+of+metabolic+fluxes.">Bacterial adaptation through distributed sensing of metabolic fluxes.</a> Mol Syst Biol. 6, 355 (2010).</li><li>Kremling, A., Bettenbrock, K., Gilles, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+global+control+of+Escherichia+coli+carbohydrate+uptake.">Analysis of global control of Escherichia coli carbohydrate uptake.</a> BMC Syst. Biol. 1, (2007).</li><li>Li, L., Liu, X., Yang, W., Xu, F., Wang, W., Feng, L., Bartlam, M., Wang, L., Rao, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+long-chain+alkane+monooxygenase+(LadA)+in+complex+with+coenzyme+FMN:+unveiling+the+long-chain+alkane+hydroxylase.">Crystal structure of long-chain alkane monooxygenase (LadA) in complex with coenzyme FMN: unveiling the long-chain alkane hydroxylase.</a> J. Mol. Biol. 376 (2), 453-465 (2008).</li><li>Lin, H. Y., Mathiszik, B., Xu, B., Enfors, S. O., Neubauer, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+the+maximum+specific+uptake+capacities+for+glucose+and+oxygen+in+glucose-limited+fed-batch+cultivations+of+Escherichia+coli.">Determination of the maximum specific uptake capacities for glucose and oxygen in glucose-limited fed-batch cultivations of Escherichia coli.</a> Biotechnol. Bioeng. 73 (5), 347-357 (2001).</li><li>Okochi, M., Kanie, K., Kurimoto, M., Yohda, M., Honda, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overexpression+of+prefoldin+from+the+hyperthermophilic+archaeum+Pyrococcus+horikoshii+OT3+endowed+Escherichia+coli+with+organic+solvent+tolerance.">Overexpression of prefoldin from the hyperthermophilic archaeum Pyrococcus horikoshii OT3 endowed Escherichia coli with organic solvent tolerance.</a> Appl. Microbiol. Biotechnol. 79 (3), 443-449 (2008).</li><li>Rehm, H. J., Reiff, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+and+occurrence+of+microbial+oxidation+of+long-chain+alkanes.">Mechanisms and occurrence of microbial oxidation of long-chain alkanes.</a> Adv. Biochem. Eng. / Biotechnol. 19, 175-215 (1981).</li><li>Rojo, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Degradation+of+alkanes+by+bacteria.">Degradation of alkanes by bacteria.</a> Environ. Microbiol. 11 (10), 2477-2490 (2009).</li><li>Van Beilen, J. B., Panke, S., Lucchini, S., Franchini, A. G., Rothlisberger, M., Witholt, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+Pseudomonas+putida+alkane-degradation+gene+clusters+and+flanking+insertion+sequences:+evolution+and+regulation+of+the+alk+genes.">Analysis of Pseudomonas putida alkane-degradation gene clusters and flanking insertion sequences: evolution and regulation of the alk genes.</a> Microbiology. 147, 1621-1630 (2001).</li><li>Wang, L., Tang, Y., Wang, S., Liu, R. L., Liu, M. Z., Zhang, Y., Liang, F. L., Feng, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+a+novel+thermophilic+Bacillus+strain+degrading+long-chain+n-alkanes.">Isolation and characterization of a novel thermophilic Bacillus strain degrading long-chain n-alkanes.</a> Extremophiles. 10 (4), 347-356 (2006).</li></ol>

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BioBrick原则被用于构造的底架的与链烷烃的降解和该工具包为单个元件的一个原则的证明得到。几个实验提出在体内和体外活性烷烃降解途径酶的测量。所提出的工作成功地演示了一些方法，这些方法可以被用来确定酶的活性和表达在宿主有机体E.大肠杆菌合适的BioBricks后实施。此外，它是示出，该BioBrick原则可以用来设计一个有机体表达蛋白质所需的链烷烃的降解，提供​​了一个...

Oil pollution is among the most serious causes of environmental contamination, and greatly affects ecosystems, businesses and communities 3. Solutions are for example required to battle the continuous oil pollution originating from the oil sands tailing waters in Alberta, Canada. During the process of oil extraction from oil sands, bitumen, a semi-solid oxidized form of oil, is removed using thermal recovery techniques that consume about 3.1 barrels of water per single barrel of oil 1. Oil contaminated process water, mainly originating from a local river, is stored in tailing ponds after bitumen extraction. A more effective recycling of process water in order to reduce the need for freshwater uptake is needed. To facilitate the bitumen extraction and to ensure that downstream sites meet water quality guidelines for the protection of aquatic ecosystems, process water treatments are rapidly evolving 3.
To treat pollution of organic compounds, bioremediation technologies employing microorganisms are presently encouraged 1. Alkanes are the most abundant family of hydrocarbons in crude oil, containing 5 to 40 carbon atoms per molecule 7, 21. Many bacteria are known to degrade alkanes of various lengths via sequential oxidation of the terminal methyl group forming first alcohols, then aldehydes and finally fatty acids 8. Within this iGEM project several enzymes from different organisms were expressed and characterized, and made available via the BioBrick standard and Registry of Standard Biological Parts.
The well-studied alkane hydroxylase system of Gordonia sp. TF6 facilitates the initial oxidation step of C5-C13 alkanes along with that of C5-C8 cycloalkanes using a minimum of four components: alkB2 (alkane 1-monooxygenase), rubA3, rubA4 (two rubredoxins) and RubB (rubredoxin reductase) 8, 21. Oxidation of long-chain alkanes (ranging from C15 up to C36) is reported to be performed by ladA, a flavoprotein alkane monooxygenase from Geobacillus thermodinitrificans NG-80-2 7, 15, 18, 22. LadA forms a catalytic complex with flavin mononucleotide (FMN) that utilizes atomic oxygen for oxidation. This results in the conversion of alkanes into the corresponding primary alkanol. The alcohols are further oxidized by alcohol and aldehyde dehydrogenases to fatty acids, which readily enter the &beta;-oxidation pathway 7, 21. A zinc-independent alcohol dehydrogenase from the thermophillic bacterium Geobacillus thermoleovorans B23 oxidizes medium-chain alkanols into their respective alkanals, using NAD+ as a cofactor 10. Aldehyde dehydrogenase from the same bacterium is able to catalyze the NAD+-dependent final step in the medium-chain oxidation 11.
In order to reduce induction costs and to maintain optimal proliferation of the bacterial system, the promoter pCaiF from E.coli was characterized. This promoter can regulate expression of the hydrocarbon degradation pathway components, and is regulated by cAMP-Crp levels, which in turn depend on glucose levels 6. At high extracellular glucose concentrations in the environment the cellular cAMP (cyclic Adenosine Mononucleotide Phosphate) level was low through the inhibition of adenylyl cyclase as a side effect of PTS mediated glucose transport. Conversely, during limitation (low glucose concentrations) the cAMP level increased and Crp bound to cAMP forming the complex, cAMP-Crp, which bound pCaiF and activated transcription of the downstream components 6, 14.
Wildtype E. coli can only tolerate moderate concentrations of hydrocarbons. To complete the toolkit, tolerance to hydrocarbons had to be addressed. Several organic solvent-tolerant bacteria are known to survive in water-solvent two-phase systems 12. Molecular components known to increase tolerance are chaperones that facilitate the correct folding of proteins. The prefoldin system from Pyrococcus horikoshii OT3, consisting of the proteins phPFD&alpha; and phPFD&beta;, was shown to increase hydrocarbon-tolerance 17.
The alkane conversion toolkit was constructed following the BioBrick principle, which is documented at the Registry of Standard Biological Parts 9. BioBricks are plasmids containing a specific functional insert that is flanked by 4 predefined restriction sites. The BioBrick inserts can be extended flexibly, allowing the construction of biological systems with new functions.

<table><tbody><tr><td>&lt;em&gt;E. coli&lt;/em&gt; K12</td><td>New England Biolabs</td><td>C2523H</td><td></td></tr><tr><td>Octane</td><td>Fluka</td><td>74822</td><td></td></tr><tr><td>Hexadecane</td><td>Fluka</td><td>52209</td><td></td></tr><tr><td>octanol-1</td><td>Fluka</td><td>95446</td><td></td></tr><tr><td>dodecanol-1</td><td>Sigma-Aldrich</td><td>126799</td><td></td></tr><tr><td>Hexane</td><td>Sigma-Aldrich</td><td>296090</td><td></td></tr><tr><td>NADH</td><td>Sigma-Aldrich</td><td>N4505</td><td></td></tr><tr><td>FMN</td><td>Sigma-Aldrich</td><td>F2253</td><td></td></tr><tr><td>MgSO4</td><td>J.T. Baker Casno</td><td>7487 889</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>T8787</td><td></td></tr><tr><td>T4 ligase</td><td>New England Biolabs</td><td>M0202L</td><td></td></tr><tr><td>Gas chromatograph</td><td></td><td></td><td></td></tr><tr><td>Cell disrupter</td><td>LA Biosystems</td><td>CD-019</td><td></td></tr><tr><td>Spectrophotometer</td><td>Amersham pharmacia</td><td>spec 2000</td><td></td></tr><tr><td>Plate reader</td><td>Tecan Group Ltd.</td><td>Magellan v7.0</td><td></td></tr><tr><td>Incubator </td><td>Innova, 44</td><td></td><td></td></tr><tr><td>BioBrickTM K398014: BBa_J23100-BBa_J61100-&lt;em&gt;alkB2&lt;/em&gt;-BBa_J61100-&lt;em&gt;rubA3&lt;/em&gt;-BBa_J61100-&lt;em&gt;rubA4&lt;/em&gt;- BBa_J61100-&lt;em&gt;rubB&lt;/em&gt;</td><td>Delft University of Technology at the department of Biotechnology or Registry of Standard Biological Parts</td><td>&lt;a href=&quot;http://partsregistry.org/Part:BBa_K398014&quot; target=&quot;_blank&quot;&gt;BBa_K398014&lt;/a&gt;</td><td>Alkane Hydroxylase System&lt;br/&gt; Resistance: Chloramphenicol </td></tr><tr><td>BioBrickTM K398027: BBa_R0040-BBa_B0034-&lt;em&gt;ladA&lt;/em&gt; </td><td>Delft University of Technology at the department of Biotechnology or Registry of Standard Biological Parts</td><td>&lt;a href=&quot;http://partsregistry.org/Part:BBa_K398027&quot; target=&quot;_blank&quot;&gt;BBa_K398027&lt;/a&gt;</td><td>ladA Protein Generator&lt;br/&gt; Resistance: Chloramphenicol </td></tr><tr><td>BioBrickTM K398018: BBa_J23100-BBa_J61101-&lt;em&gt;ADH&lt;/em&gt; </td><td>Delft University of Technology at the department of Biotechnology or Registry of Standard Biological Parts</td><td>&lt;a href=&quot;http://partsregistry.org/Part:BBa_K398018&quot; target=&quot;_blank&quot;&gt;BBa_K398018&lt;/a&gt;</td><td>ADH generator&lt;br/&gt; Resistance: Chloramphenicol </td></tr><tr><td>BioBrickTM K398030: BBa_R0040-BBa_B0034-&lt;em&gt;ALDH&lt;/em&gt; </td><td>Delft University of Technology at the department of Biotechnology or Registry of Standard Biological Parts</td><td>&lt;a href=&quot;http://partsregistry.org/Part:BBa_K398030&quot; target=&quot;_blank&quot;&gt;BBa_K398030&lt;/a&gt;</td><td>ALDH generator&lt;br/&gt; Resistance: Chloramphenicol </td></tr><tr><td>BioBrickTM K398326:&lt;em&gt; pCaiF&lt;/em&gt; </td><td>Delft University of Technology at the department of Biotechnology or Registry of Standard Biological Parts</td><td>&lt;a href=&quot;http://partsregistry.org/Part:BBa_K398326&quot; target=&quot;_blank&quot;&gt;BBa_K398326&lt;/a&gt;</td><td>pCaiF promoter&lt;br/&gt; Resistance: Chloramphenicol </td></tr><tr><td>BioBrickTM K398331:&lt;em&gt; pCaiF&lt;/em&gt;-BBa_B0032-BBa_I13401</td><td>Delft University of Technology at the department of Biotechnology or Registry of Standard Biological Parts</td><td>&lt;a href=&quot;http://partsregistry.org/Part:BBa_K398331&quot; target=&quot;_blank&quot;&gt;BBa_K398331&lt;/a&gt;</td><td>pCaiF measurement device&lt;br/&gt; Resistance: Chloramphenicol </td></tr><tr><td>BioBrickTM K398406: BBa_J23002-BBa_J61107-&lt;em&gt;phPFD&lt;/em&gt;&lt;em&gt;&amp;alpha;&lt;/em&gt;-BBa_J61107- </td><td>Delft University of Technology at the department of Biotechnology or Registry of Standard Biological Parts</td><td>&lt;a href=&quot;http://partsregistry.org/Part:BBa_K398406&quot; target=&quot;_blank&quot;&gt;BBa_K398406&lt;/a&gt;</td><td>Solvent tolerance cluster&lt;br/&gt; Resistance: Chloramphenicol </td></tr></tbody></table>

a toolkit to enable hydrocarbon conversion in aqueous environments

这项工作提出了一个工具包，使烷烃转化大肠杆菌 ，并提出证明其适用的原则。该工具包包括多个标准的可互换的部分（BioBricks）的9解决烷烃转化，调节基因的表达和生存有毒碳氢化合物丰富的环境。 E.实施三个步骤的烷烃降解途径大肠杆菌介质和长链烷烃的转化率，以使各自的链烷醇，烷醛和最终链烷酸。后者则是通过本机的β-氧化途径代谢。为了方便的中链烷烃的（C5-C13）和环烷烃（C5-C8），4个基因（alkB2，rubA3 rubA4和擦除 ）的烷烃羟化酶系统从大头属的氧化。 TF6 8,21转化到大肠杆菌大肠杆菌 。为转换的长链烷烃（C15-C36）， 芽孢杆菌热拉达基因的实施。对于需要采取进一步措施的退化过程，ADH和ALDH（G.热 ）的10,11。活性测定采用静息细胞检测。对于每个氧化工序中，酶的活性进行了观察。 为了优化流程的效率，表达诱导低血糖条件下基板调节的启动，pCaiF，。 pCaiF是在E.大肠杆菌 K12和调节非葡萄糖碳源的降解中所涉及的基因的表达。 使用的工具包-针对生存的最后一部分-溶剂耐受的基因，PhPFDα和β，无论是从激烈热horikoshii OT3。有机溶剂可以诱导细胞应激和下降的生存能力产生负面affectin克蛋白质折叠。作为分子伴侣，PhPFDα和β改善蛋白质的折叠过程中， 例如，链烷烃的存在下。所示的一种改进的烃类公差在培养基中的增加的生长率（高达70％）10％n-己烷的存在导致这些基因的表达进行了观察。 总之，结果表明，该工具包能够E.大肠杆菌转换和容忍在水环境中的碳氢化合物。因此，它代表了一个可持续发展的解决方案，油整治利用合成生物学的方法的第一步。

Alkane conversion
The activity of the three oxidation steps from the alkane to the respective fatty acid was evaluated using resting cell assays and enzyme activity measurements. The results are presented following the pathway reactions (1) alkane hydroxylase, (2) alcohol dehydrogenase and (3) aldehyde dehydrogenase. 
For the first step, different plasmids were constructed for medium and long-chain alkanes. The plasmid <a href="http://partsregistry.org/Part:BBa_K398014" target="_blank">BBa_...

Watch this Scientific Journal Video about A Toolkit to Enable Hydrocarbon Conversion in Aqueous Environments at JoVE.com

在水环境中实现碳氢化合物转化的工具包生物工程|视频

A Toolkit to Enable Hydrocarbon Conversion in Aqueous Environments

一个可持续发展的自动调节修复石油污染的细菌系统的设计采用标准的可互换的DNA部分（BioBricks）。一个工程<em&gt; E.大肠杆菌</em&gt;菌株用于在有毒的水溶液环境中，通过β-氧化降解烷烃。来自不同物种的酶显示烷烃的降解活性。此外，增加的耐受性<em&gt; N</em&gt;己烷，通过引入从烷烃耐受菌的基因来实现。

一个工具包，使水环境中的烃类转化

This work puts forward a toolkit that enables the conversion of alkanes by Escherichia coli and presents a proof of principle of its applicability. The ...

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Alkanes and Cycloalkanes

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14.0K 次观看.  Delft University of Technology. 一个可持续发展的自动调节修复石油污染的细菌系统的设计采用标准的可互换的DNA部分（BioBricks）。一个工程 E.大肠杆菌菌株用于在有毒的水溶液环境中，通过β-氧化降解烷烃。来自不同物种的酶显示烷烃的降解活性。此外，增加的耐受性 N己烷，通过引入从烷烃耐受菌的基因来实现。

视频: A Toolkit to Enable Hydrocarbon Conversion in Aqueous Environments

Biomass Conversion to Produce Hydrocarbon Liquid Fuel Via Hot-vapor Filtered Fast Pyrolysis and Catalytic Hydrotreating

Lignocellulosic biomass conversion to produce biofuels has received significant attention because of the quest for a replacement for fossil fuels. Among the various thermochemical and biochemical routes, fast pyrolysis followed by catalytic hydrotreating is considered to be a promising near-term opportunity. This paper reports on experimental methods used 1) at the National Renewable Energy Laboratory (NREL) for fast pyrolysis of lignocellulosic biomass to produce bio-oils in a fluidized-bed reactor and 2) at Pacific Northwest National Laboratory (PNNL) for catalytic hydrotreating of bio-oils in a two-stage, fixed-bed, continuous-flow catalytic reactor. The configurations of the reactor systems, the operating procedures, and the processing and analysis of feedstocks, bio-oils, and biofuels are described in detail in this paper. We also demonstrate hot-vapor filtration during fast pyrolysis to remove fine char particles and inorganic contaminants from bio-oil. Representative results showed successful conversion of biomass feedstocks to fuel-range hydrocarbon biofuels and, specifically, the effect of hot-vapor filtration on bio-oil production and upgrading. The protocols provided in this report could help to generate rigorous and reliable data for biomass pyrolysis and bio-oil hydrotreating research.

Experimental methods for fast pyrolysis of lignocellulosic biomass to produce bio-oils and for the catalytic hydrotreating of bio-oils to produce fuel range hydrocarbons are presented. Hot-vapor filtration during fast pyrolysis to remove fine char particles and inorganic contaminants from bio-oil was also assessed.

生物质转化为油气生产液体燃料通过热蒸汽过滤快速热解和催化加氢

Lignocellulosic biomass conversion to produce biofuels has received significant attention because of the quest for a replacement for fossil fuels. Among ...

科研

生物化学

Laboratory Production of Biofuels and Biochemicals from a Rapeseed Oil through Catalytic Cracking Conversion

The work is based on a reported study which investigates the processability of canola oil (bio-feed) in the presence of bitumen-derived heavy gas oil (HGO) for production of transportation fuels through a fluid catalytic cracking (FCC) route. Cracking experiments are performed with a fully automated reaction unit at a fixed weight hourly space velocity (WHSV) of 8 hr-1, 490-530 &#176;C, and catalyst/oil ratios of 4-12 g/g. When a feed is in contact with catalyst in the fluid-bed reactor, cracking takes place generating gaseous, liquid, and solid products. The vapor produced is condensed and collected in a liquid receiver at -15 &#176;C. The non-condensable effluent is first directed to a vessel and is sent, after homogenization, to an on-line gas chromatograph (GC) for refinery gas analysis. The coke deposited on the catalyst is determined in situ by burning the spent catalyst in air at high temperatures. Levels of CO2 are measured quantitatively via an infrared (IR) cell, and are converted to coke yield. Liquid samples in the receivers are analyzed by GC for simulated distillation to determine the amounts in different boiling ranges, i.e., IBP-221 &#176;C (gasoline), 221-343 &#176;C (light cycle oil), and 343 &#176;C+ (heavy cycle oil). Cracking of a feed containing canola oil generates water, which appears at the bottom of a liquid receiver and on its inner wall. Recovery of water on the wall is achieved through washing with methanol followed by Karl Fischer titration for water content. Basic results reported include conversion (the portion of the feed converted to gas and liquid product with a boiling point below 221 &#176;C, coke, and water, if present) and yields of dry gas (H2-C2's, CO, and CO2), liquefied petroleum gas (C3-C4), gasoline, light cycle oil, heavy cycle oil, coke, and water, if present.

This paper presents an experimental method to produce biofuels and biochemicals from canola oil mixed with a fossil-based feed in the presence of a catalyst at mild temperatures. Gaseous, liquid, and solid products from a reaction unit are quantified and characterized. Conversion and individual product yields are calculated and reported.

通过催化裂化转换从菜籽油生物燃料和生物化学实验室生产

The work is based on a reported study which investigates the processability of canola oil (bio-feed) in the presence of bitumen-derived heavy gas oil ...

化学

Synthesis and Testing of Supported Pt-Cu Solid Solution Nanoparticle Catalysts for Propane Dehydrogenation

A convenient method for the synthesis of bimetallic Pt-Cu catalysts and performance tests for propane dehydrogenation and characterization are demonstrated here. The catalyst forms a substitutional solid solution structure, with a small and uniform particle size around 2 nm. This is realized by careful control over the impregnation, calcination, and reduction steps during catalyst preparation and is identified by advanced in situ synchrotron techniques. The catalyst propane dehydrogenation performance continuously improves with increasing Cu:Pt atomic ratio.

A convenient method for the synthesis of 2 nm supported bimetallic nanoparticle Pt-Cu catalysts for propane dehydrogenation is reported here. In situ synchrotron X-ray techniques allow for the determination of the catalyst structure, which is typically unobtainable using laboratory instruments.

用于丙烷脱氢的负载型Pt-Cu固溶体纳米粒子催化剂的合成与测试

A convenient method for the synthesis of bimetallic Pt-Cu catalysts and performance tests for propane dehydrogenation and characterization are ...

Utilization of Stop-flow Micro-tubing Reactors for the Development of Organic Transformations

A new reaction screening technology for organic synthesis was recently demonstrated by combining elements from both continuous micro-flow and conventional batch reactors, coined stop-flow micro-tubing (SFMT) reactors. In SFMT, chemical reactions that require high pressure can be screened in parallel through a safer and convenient way. Cross-contamination, which is a common problem in reaction screening for continuous flow reactors, is avoided in SFMT. Moreover, the commercially available light-permeable micro-tubing can be incorporated into SFMT, serving as an excellent choice for light-mediated reactions due to a more effective uniform light exposure, compared to batch reactors. Overall, the SFMT reactor system is similar to continuous flow reactors and more superior than batch reactors for reactions that incorporate gas reagents and/or require light-illumination, which enables a simple but highly efficient reaction screening system. Furthermore, any successfully developed reaction in the SFMT reactor system can be conveniently translated to continuous-flow synthesis for large scale production.

A protocol for organic reaction screening using stop-flow micro-tubing (SFMT) reactors employing gaseous reactants and/or visible-light mediated reactions is presented.

停止流动微管反应器在有机转化发展中的应用

A new reaction screening technology for organic synthesis was recently demonstrated by combining elements from both continuous micro-flow and conventional ...

Isolation, Propagation, and Identification of Bacterial Species with Hydrocarbon Metabolizing Properties from Aquatic Habitats

Hydrocarbon pollutants are recalcitrant to degradation and their accumulation in the environment is toxic to all life forms. Bacteria encode numerous catalytic enzymes and are naturally capable of metabolizing hydrocarbons. Scientists harness biodiversity in aquatic ecosystems to isolate bacteria with biodegradation and bioremediation potential. Such isolates from the environment provide a rich set of metabolic pathways and enzymes, which can be further utilized to scale up the degradation process at an industrial scale. In this article, we outline the general process of isolation, propagation, and identification of bacterial species from aquatic habitats and screen their ability to utilize hydrocarbons as the sole carbon source in vitro using simple techniques. The present protocol describes the isolation of various bacterial species and their subsequent identification using the 16S rRNA analysis. The protocol also presents steps for characterizing the hydrocarbon degrading potential of bacterial isolates. This protocol will be useful for researchers trying to isolate bacterial species from environmental habitats for their biotechnological applications.

We present the process of isolating, propagating, and characterizing hydrocarbon-degrading bacteria from aquatic habitats. The protocol outlines bacterial isolation, identification by the 16S rRNA method, and testing of their hydrocarbon-degrading potential. This article would help researchers in characterizing microbial biodiversity in environmental samples, and specifically screen for microbes with bioremediation potential.

从水生栖息地分离、繁殖和鉴定具有碳氢化合物代谢特性的细菌种类

Hydrocarbon pollutants are recalcitrant to degradation and their accumulation in the environment is toxic to all life forms. Bacteria encode numerous ...

环境科学

Tuning the Acidity of Pt/ CNTs Catalysts for Hydrodeoxygenation of Diphenyl Ether

We herein present a method for the synthesis of HNbWO6, HNbMoO6, HTaWO6 solid acid nanosheet modified Pt/CNTs. By varying the weight of various solid acid nanosheets, a series of Pt/xHMNO6/CNTs with different solid acid compositions (x = 5, 20 wt%; M = Nb, Ta; N = Mo, W) have been prepared by carbon nanotube pretreatment, protonic exchange, solid acid exfoliation, aggregation and finally Pt particles impregnation. The Pt/xHMNO6/CNTs are characterized by X-ray diffraction, scanning electron microscopy, transmission electron microscopy and NH3-temperature programmed desorption. The study revealed that HNbWO6 nanosheets were attached on CNTs, with some edges of the nanosheets being bent in shape. The acid strength of the supported Pt catalysts increases in the following order: Pt/CNTs &#60; Pt/5HNbWO6/CNTs &#60; Pt/20HNbMoO6/CNTs &#60; Pt/20HNbWO6/CNTs &#60; Pt/20HTaWO6/CNTs. In addition, the catalytic hydroconversion of lignin-derived model compound: diphenyl ether using the synthesized Pt/20HNbWO6 catalyst has been investigated.

A protocol for the synthesis of HNbWO6, HNbMoO6, HTaWO6 solid acid nanosheet modified Pt/CNTs is presented.

调整二苯醚氢氧化的Pt/CNT催化剂的酸度

We herein present a method for the synthesis of HNbWO6, HNbMoO6, HTaWO6 solid acid nanosheet modified Pt/CNTs. By varying the weight of various solid acid ...

Qualitative Characterization of the Aqueous Fraction from Hydrothermal Liquefaction of Algae Using 2D Gas Chromatography with Time-of-flight Mass Spectrometry

Two-dimensional gas chromatography coupled with time-of-flight mass spectrometry is a powerful tool for identifying and quantifying chemical components in complex mixtures. It is often used to analyze gasoline, jet fuel, diesel, bio-diesel and the organic fraction of bio-crude/bio-oil. In most of those analyses, the first dimension of separation is non-polar, followed by a polar separation. The aqueous fractions of bio-crude and other aqueous samples from biofuels production have been examined with similar column combinations. However, sample preparation techniques such as derivatization, solvent extraction, and solid-phase extraction were necessary prior to analysis. In this study, aqueous fractions obtained from the hydrothermal liquefaction of algae were characterized by two-dimensional gas chromatography coupled with time-of-flight mass spectrometry without prior sample preparation techniques using a polar separation in the first dimension followed by a non-polar separation in the second. Two-dimensional plots from this analysis were compared with those obtained from the more traditional column configuration. Results from qualitative characterization of the aqueous fractions of algal bio-crude are discussed in detail. The advantages of using a polar separation followed by a non-polar separation for characterization of organics in aqueous samples by two-dimensional gas chromatography coupled with time-of-flight mass spectrometry are highlighted.

A two-dimensional gas chromatography-time-of-flight mass spectrometry method is described for characterization of the aqueous fraction of bio-crude produced from hydrothermal liquefaction of algae. This protocol can also be employed to analyze the aqueous fraction of liquid products from fast pyrolysis, catalytic fast pyrolysis, catalytic deoxygenation and hydro-treating.

从藻类水热液化使用二维气相色谱时间飞行质谱法水提取部分的定性鉴定

Two-dimensional gas chromatography coupled with time-of-flight mass spectrometry is a powerful tool for identifying and quantifying chemical components in ...

生物工程

Catalytic Reactions at Amine-Stabilized and Ligand-Free Platinum Nanoparticles Supported on Titania During Hydrogenation of Alkenes and Aldehydes

Ligands like amines are used in the colloidal synthesis approach to protect platinum nanoparticles (Pt NP&#39;s) from agglomeration. Normally, ligands like amines are removed by diverse pre-treatment procedures before use in heterogeneous catalysis as amines are considered as a catalyst poison. However, a possible beneficial influence of these surface modifiers on hydrogenation reactions, which is known from spectator species on metal surfaces, is often neglected.
Therefore, amine-stabilized Pt nanoparticles supported by titania (P25) were used without any pre-treatment in order to elucidate a possible influence of the ligand in liquid phase hydrogenation reactions. The catalytic activity of amine-stabilized Pt nanoparticles of two different sizes was investigated in a double-walled stirring tank reactor at 69 &#176;C to 130 &#176;C and 1 atm hydrogen pressure. The conversion of cyclohexene to cyclohexane was determined by gas chromatography (GC) and was compared to ligand-free Pt particles. All catalysts were checked before and after reaction by transmission electron spectroscopy (TEM) and X-ray photoelectron spectroscopy (XPS) for possible changes in size, shape, and ligand shell. The hydrogenation of cyclohexene in liquid phase revealed a higher conversion for amine-stabilized Pt nanoparticles on titania than the ligand-free particles. The hydrogenation of 5-methylfurfural (5-MF) was chosen for a further test reaction, since the hydrogenation of &#945;, &#946;-unsaturated aldehydes is more complex and exhibits various reaction paths. However, XPS and infrared spectroscopy (IR) proved that 5-MF acts as catalyst poison at the given reaction conditions.

This protocol shows a convenient method for comparing the catalytic properties of supported platinum catalysts, synthesized by deposition of nanosized colloids or by impregnation. The hydrogenation of cyclohexene serves as a model reaction to determine the catalytic activity of the catalysts.

烯烃和醛加氢过程中负载在二氧化钛上的胺稳定和无配体铂纳米颗粒的催化反应

Ligands like amines are used in the colloidal synthesis approach to protect platinum nanoparticles (Pt NP&#39;s) from agglomeration. Normally, ligands ...

通过超声辅助植物油酯交换合成生物柴油

Ultrasonic-Assisted Preparation of Biodiesel Products from Vegetable Oils

Utilizing vegetable oil as a sustainable feedstock, this study presents an innovative approach to ultrasonic-assisted transesterification for biodiesel synthesis. This alkaline-catalyzed procedure harnesses ultrasound as a potent energy input, facilitating the rapid conversion of extra virgin olive oil into biodiesel. In this demonstration, the reaction is run in an ultrasonic bath under ambient conditions for 15 min, requiring a 1:6 molar ratio of extra virgin olive oil to methanol and a minimum amount of KOH as the catalyst. The physiochemical properties of biodiesel are also reported. Emphasizing the remarkable advantages of ultrasonic-assisted transesterification, this method demonstrates notable reductions in reaction and separation times, achieving near-perfect purity (~100%), high yields, and negligible waste generation. Importantly, these benefits are achieved within a framework that prioritizes safety and environmental sustainability. These compelling findings underscore the effectiveness of this approach in converting vegetable oil into biodiesel, positioning it as a viable option for both research and practical applications.

作者聚焦:采用绿色化学原则安全可持续地合成生物柴油

A safe ultrasonic-assisted transesterification method for vegetable oils using an alkaline catalyst is presented here. The method is rapid and efficient for preparing pure biodiesel products.

超声辅助制备植物油生物柴油产品

Utilizing vegetable oil as a sustainable feedstock, this study presents an innovative approach to ultrasonic-assisted transesterification for biodiesel ...

一个工具包，使水环境中的烃类转化

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通过催化裂化转换从菜籽油生物燃料和生物化学实验室生产

用于丙烷脱氢的负载型Pt-Cu固溶体纳米粒子催化剂的合成与测试

停止流动微管反应器在有机转化发展中的应用

从水生栖息地分离、繁殖和鉴定具有碳氢化合物代谢特性的细菌种类

调整二苯醚氢氧化的Pt/CNT催化剂的酸度

从藻类水热液化使用二维气相色谱时间飞行质谱法水提取部分的定性鉴定

烯烃和醛加氢过程中负载在二氧化钛上的胺稳定和无配体铂纳米颗粒的催化反应

超声辅助制备植物油生物柴油产品

超声辅助制备植物油生物柴油产品