Cavity-Enhanced Raman Spectroscopy in the Biosciences: In Situ, Multicomponent, and Isotope Selective Gas Measurements To Study Hydrogen Production and Consumption by Escherichia coli.
Cavity-Enhanced Raman Spectroscopy in the Biosciences: In Situ, Multicomponent, and Isotope Selective Gas Measurements To Study Hydrogen Production and Consumption by Escherichia coli.
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DOI：
10.1021/acs.analchem.6b04924
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发表时间：
2017-01
影响因子：
7.4
通讯作者：
Thomas W Smith;M. Hippler
中科院分区：
文献类型：
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作者：
Thomas W Smith;M. Hippler
最近，我们介绍了腔增强拉曼光谱（CERS）与光反馈连续二极管激光器作为一个敏感的分析工具。在这里，我们报告的技术和它的第一个应用在生物科学中的改进，在原位，多组分，同位素选择性气体测量研究大肠杆菌的氢的生产和消费。在厌氧条件下，培养物生长在丰富的培养基中补充d-葡萄糖或甘油产生H2，同时消耗一些it.By引入D2的顶空，氢的生产和消耗可以分开，由于同位素异构体的独特的光谱特征。确定了具有明显不同的H2和CO2生产和D2消耗的动力学制度的不同阶段。一些消耗的D2通过与溶剂的H/D交换转化回H2。HD仅作为次要组分形成。这反映了氢化酶活性位点处的H/D交换与重组速率相比是快速的，在分子形成后发生HD的快速重新捕获，或者D2氧化和质子还原发生的活性位点被物理分离。而在葡萄糖补充的培养物中，添加D2导致产生的H2增加，而CO2的产量保持不变;对于甘油，添加D2不仅导致H2产量增加，而且还显著增加CO2产量，反映了对发酵途径的影响。CO的加入被发现完全抑制H2的产生，并显着减少D2氧化，表明至少有一些作用O2耐受Hyd-I在D2消费。
Recently we introduced cavity-enhanced Raman spectroscopy (CERS) with optical feedback cw-diode lasers as a sensitive analytical tool. Here we report improvements made on the technique and its first application in the biosciences for in situ, multicomponent, and isotope selective gas measurements to study hydrogen production and consumption by Escherichia coli. Under anaerobic conditions, cultures grown on rich media supplemented with d-glucose or glycerol produce H2 and simultaneously consume some of it. By introducing D2 in the headspace, hydrogen production and consumption could be separated due to the distinct spectroscopic signatures of isotopomers. Different phases with distinctly different kinetic regimes of H2 and CO2 production and D2 consumption were identified. Some of the D2 consumed is converted back to H2 via H/D exchange with the solvent. HD was formed only as a minor component. This reflects either that H/D exchange at hydrogenase active sites is rapid compared to the rate of recombination, rapid recapture of HD occurs after the molecule is formed, or that the active sites where D2 oxidation and proton reduction occur are physically separated. Whereas in glucose supplemented cultures, addition of D2 led to an increase in H2 produced, while the yield of CO2 remained unchanged; with glycerol, addition of D2 led not only to increased yields of H2, but also significantly increased CO2 production, reflecting an impact on fermentation pathways. Addition of CO was found to completely inhibit H2 production and significantly reduce D2 oxidation, indicating at least some role for O2-tolerant Hyd-1 in D2 consumption.