Department of Chemistry, University of Sheffield , Sheffield S3 7HF, United Kingdom.
Anal Chem. 2017 Feb 7;89(3):2147-2154. doi: 10.1021/acs.analchem.6b04924. Epub 2017 Jan 23.
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 H and simultaneously consume some of it. By introducing D 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 H and CO production and D consumption were identified. Some of the D consumed is converted back to H 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 D oxidation and proton reduction occur are physically separated. Whereas in glucose supplemented cultures, addition of D led to an increase in H produced, while the yield of CO remained unchanged; with glycerol, addition of D led not only to increased yields of H, but also significantly increased CO production, reflecting an impact on fermentation pathways. Addition of CO was found to completely inhibit H production and significantly reduce D oxidation, indicating at least some role for O-tolerant Hyd-1 in D consumption.
最近，我们介绍了采用光学反馈连续波二极管激光器的腔增强拉曼光谱（CERS）作为一种灵敏的分析工具。在这里，我们报告了对该技术的改进及其在生物科学中的首次应用，用于原位、多组分和同位素选择性气体测量，以研究大肠杆菌的氢气产生和消耗。在厌氧条件下，在富含营养的培养基上生长并添加 d-葡萄糖或甘油的培养物会产生 H 并同时消耗一部分 H。通过在顶空引入 D，可以由于同位异构体的不同光谱特征来分离氢气产生和消耗。确定了具有明显不同的 H 和 CO 产生以及 D 消耗动力学阶段的不同阶段。一些消耗的 D 通过与溶剂的 H/D 交换重新转化为 H。仅形成少量 HD。这反映了氢酶活性位点处的 H/D 交换与重组速率相比是快速的，或者 D 氧化和质子还原发生的活性位点在物理上是分开的，或者形成分子后 HD 迅速重新捕获。在添加葡萄糖的培养物中，添加 D 会导致产生的 H 增加，而 CO 的产率保持不变；而在添加甘油的培养物中，添加 D 不仅会导致 H 的产率增加，而且还会显著增加 CO 的产生，这反映了对发酵途径的影响。发现添加 CO 会完全抑制 H 的产生并显著降低 D 的氧化，表明至少在某些情况下 O-耐受型 Hyd-1 参与了 D 的消耗。
