乙醇应用于厌氧消化中直接种间电子传递的构建及其调控机制

Direct Interspecies electron transfer (DIET), a novel pathway for interspecies electron and energy exchange, is capable of replacing the traditional interspecies hydrogen transfer (IHT) as well as effectively maintaining the balance between anaerobic acidogenesis and methanogenesis. However, up to now, only Geobacter species that are usually not abundant in most of conventional anaerobic digesters have been confirmed to proceed DIET. Moreover, the available substrates for Geobacter species to proceed DIET-based methanogenesis are only limited small-molecule organic acids and alcohols. Previous studies showed that, stimulating the conventional methanogenic communities with ethanol could significantly enrich Geobacter species and proceed DIET-based methanogenesis to utilize the dysoxidizable organic acids in thermodynamics. It is suggested that, Geobacter species are stimulated to develop the novel pathway for the metabolism of organic acids when ethanol is served as the co-metabolism substrate. Based on this assumption, a study will be conducted in this proposal to explore the application of ethanol in establishment of DIET during anaerobic digestion and its adjustment mechanism, including the following contents: (1) Roles of enriching DIET-based syntrophs with ethanol; (2) Mechanisms of establishing the DIET-based methanogenic pathway; (3) Roles of ethanol-type fermentation to the DIET-based methanogenesis and their adjustment strategies. We expect to reveal the mechanisms of establishing DIET-based methanogenic pathway with ethanol, in order to achieve the better application of DIET during anaerobic digestion.

直接种间电子传递(DIET)是能取代种间氢气传递(IHT)，并有效维持厌氧的产酸与产甲烷代谢平衡的种间电子交换新途径。然而，目前唯一被证实驱动DIET的Geobacter在常规厌氧消化系统匮乏，且由其驱动的DIET仅能利用有限几种小分子有机酸和醇。前期研究表明，采用乙醇刺激常规产甲烷群落，能显著富集Geobacter，推动DIET利用热力学上较难氧化的有机酸。我们推测这是由于乙醇作为共代谢基质，刺激Geobacter开辟有机酸降解新途径。基于此，本课题拟开展乙醇应用于厌氧消化中DIET的构建及其调控机制研究。主要研究内容包括：(1)乙醇对于DIET微生物的富集规律；(2)乙醇对于DIET产甲烷路径的构建机制；(3)乙醇型发酵对于DIET产甲烷的作用规律及调控策略。通过以上研究，期待揭示乙醇对于DIET产甲烷路径的构建机制，为DIET更好地应用于厌氧消化提供技术支持。

现有的厌氧消化工程普遍技术陈旧，其核心原理仍停留在以H2扩散为主导的传统水解酸化-产甲烷路径，面临“尴尬”局面——发酵速率慢、有机质转化效率低、系统稳定性差。直接种间电子传递(DIET)产甲烷突破了复杂有机物水解的速率约束及有机物分解产乙酸的热力学屏障，开辟了厌氧消化新路径。然而，构建DIET产甲烷需借助铁还原菌及其胞外电子传递作用。但常规厌氧消化系统中铁还原菌丰度不高，导致DIET产甲烷不明显。当进料中存在少量乙醇时，铁还原菌富集明显，且与产甲烷菌快速聚集并形成导电颗粒污泥，推动DIET产甲烷发生。为进一步探索乙醇构建DIET产甲烷的微生物原理及调控方法，本课题开展了如下研究内容：①乙醇对于DIET微生物的富集规律；②乙醇对于DIET产甲烷路径的构建机制；③乙醇型发酵对于DIET产甲烷的作用规律及调控策略。结果显示：当进料中存在少量乙醇时，铁还原菌丰度提高近14倍，污泥电导率提高2.8-3.7倍，甲烷产量提高22-45%。宏基因组测序发现乙醇可刺激铁还原菌胞内ATP合成的关键酶——琥珀酸脱氢酶，进而驱动醌池(UQ/UQH2)反应，加快H+跨膜梯度的形成，以产生更多的ATP，促使铁还原菌分泌胞外导电纳米菌丝，与产甲烷菌形成DIET。为维持乙醇刺激效果持久，尝试进料自身产乙醇，即在两相厌氧的水解酸化段，通过控制发酵pH或投加酵母，实现部分进料定向发酵产乙醇，并耦合铁/碳等导电材料充当电子“导管”，形成乙醇型发酵预处理耦合铁/碳强化的两相厌氧工艺。该工艺在处理餐厨垃圾/剩余污泥效果尤为显著，提高餐厨垃圾甲烷产量40%以上，剩余污泥有机质转化效率从150-170 mL/g-VSS提升至233.5 mL/g-VSS。本课题揭示了乙醇构建DIET产甲烷的微生物原理，形成了乙醇构建DIET产甲烷的调控方法，有效提高厌氧消化效率和稳定，并成功应用于实际工程。