Combined carbon, hydrogen, and clumped isotope fractionations reveal differential reversibility of hydrogenotrophic methanogenesis in laboratory cultures
Combined carbon, hydrogen, and clumped isotope fractionations reveal differential reversibility of hydrogenotrophic methanogenesis in laboratory cultures
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DOI：
10.1016/j.gca.2022.07.027
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发表时间：
2022-08
影响因子：
5
通讯作者：
Jeemin H. Rhim;S. Ono
中科院分区：
文献类型：
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作者：
Jeemin H. Rhim;S. Ono
稳定同位素分析已被广泛应用于甲烷的来源识别。然而，自然环境中微生物甲烷的同位素(13C/12C和D/H)和同位素(13CH3D和12CH2D2)特征往往与实验室培养中的不同，实验室培养通常在最佳条件下生长产甲烷菌。生长阶段和氢(H2)浓度被认为是控制氢遗传营养产甲烷所产生的甲烷的同位素组成的因素，但它们对碳、氢和双取代“簇状”同位素体系之间的关系的影响尚未在定量框架内进行评估。在这里，我们使用分批和补料分批系统，在不同的生长阶段和不同的H2混合比例(82℃或60°C，80%或25%H2；甲烷热营养杆菌str)下，实验研究了高温嗜水遗传营养(二氧化碳还原)产甲烷菌产生的甲烷的整体(δ13C和δD)和块状(Δ13CH3D)同位素组成。ΔH在65°C和20、5或1.6%H2下)。我们观察到了大范围(从18到63‰)的碳同位素分馏，在生长后期观察到了更大的值，这与之前的观察一致。相比之下，氢同位素分馏保持相对恒定，为-317±12.25‰。在与m的实验中观察到线性增长。随着菌体密度的增加，气态H_2在液体介质中的溶解成为速率极限。因此，低浓度(和未饱和)的溶解H_2可以解释生长后期碳同位素分馏增加的原因。在整个实验过程中，δD和Δ13CH3D的值表明偏离了平衡。随着细胞密度的增加和溶解H_2的减少，Δ13CH3D减少(进一步偏离平衡)，与以前模型的预期相反。我们的同位素流动网络模型再现了当最后一个H-加成步骤比前三个H-加成步骤(直到CH3-COM)可逆性较差时观察到的趋势。在这个微分可逆性模型中，碳、氢和簇状同位素的分馏在很大程度上受前三个氢加成步骤在高氢气浓度下的可逆性控制，最后一个氢加成步骤在低氢气浓度下变得重要。模型中考虑了较大的(Δ6≥)二次聚集动力学同位素效应，再现了‰13CH3D值的耗竭程度和下降趋势。这项研究强调了整体和块状同位素分析相结合的优势，以及在使用同位素分析更好地了解产甲烷代谢和甲烷循环过程时，生理因素(生长阶段)和能量可获得性(溶解H_2浓度)的重要性。
Stable isotope analysis has been widely used to aid the source identification of methane. However, the isotopic (13C/12C and D/H) and isotopologue (13CH3D and12CH2D2) signatures of microbial methane in natural environments are often different from those in laboratory cultures in which methanogens are typically grown under optimal conditions. Growth phase and hydrogen (H2) concentration have been proposed as factors controlling the isotopic compositions of methane produced via hydrogenotrophic methanogenesis, but their effects on the relationship among carbon, hydrogen and doubly-substituted “clumped” isotopologue systems have not been assessed in a quantitative framework. Here we experimentally investigate the bulk (δ13C and δD) and clumped (Δ13CH3D) isotopologue compositions of methane produced by hyperthermophilic hydrogenotrophic (CO2-reducing) methanogens using batch and fed-batch systems at different growth phases and H2mixing ratios (Methanocaldococcusbathoardescensat 82 or 60 °C and on 80 or 25 % H2; Methanothermobacter thermautotrophicus str. ΔH at 65 °C and on 20, 5 or 1.6 % H2). We observed a large range (from 18 to 63 ‰) of carbon isotope fractionations, with larger values observed during later growth phase, consistent with previous observations. In contrast, hydrogen isotope fractionations remained relatively constant at –317 ± 25 ‰. Linear growth was observed for experiments withM. bathoardescens, suggesting that dissolution of gaseous H2into liquid media became the rate limit as cell density increased. Accordingly, the low (and undersaturated) dissolved H2concentrations can explain the increased carbon isotope fractionations during the later growth phase. The δD and Δ13CH3D values indicated departure from equilibrium throughout experiments. As the cell density increased and dissolved H2decreased, Δ13CH3D decreased (further departure from equilibrium), contrary to expectations from previous models. Our isotopologue flow network model reproduced the observed trends when the last H-addition step is less reversible relative to the first three H-addition steps (up to CH3-CoM). In this differential reversibility model, carbon, hydrogen and clumped isotopologue fractionations are largely controlled by the reversibility of the first three H-addition steps under high H2concentrations; the last H-addition step becomes important under low H2. The magnitude of depletion and decreasing trend in Δ13CH3D values were reproduced when a large (≥6 ‰) secondary clumped kinetic isotope effect was considered in the model. This study highlights the advantage of combined bulk and clumped isotope analyses and the importance of physiological factors (growth phase) and energy availability (dissolved H2concentration) when using isotope analyses to better understand methanogenic metabolisms and methane cycling processes.