氨基酸及其对映体对污泥厌氧消化产甲烷的影响及作用机制研究

As an important intermediate product in sludge anaerobic digestion process, amino acids have two configurations, L and D, which are enantiomers of each other. Pretreatment (such as alkali treatment, heat treatment, etc.) are commonly used to strengthen the sludge anaerobic digestion process. Pretreatment will cause the protein hydrolytic amino acid racemization, and form the L-amino acids and D-amino acids. However, there are few reports about the production and distribution of amino acids and their enantiomers, as well as their influence on the anaerobic digestion process. The research objectives of this project mainly include the study of the generation and distribution of amino acids and their enantiomers in the process of protein hydrolysis, and the further investigation of the effects and mechanisms of amino acids and their enantiomers on the anaerobic digestion process. The key process and mechanism of anaerobic digestion are identified by studying each stage of anaerobic digestion. The effects and mechanisms are investigated in terms of key enzymes activities, transcriptional expression of key genes, changes in substrate characteristics and microbial community structure. The research results will be beneficial to further understand the role of amino acids and their enantiomers in the anaerobic digestion process, and provide a new perspective and method for the regulation of anaerobic digestion.

氨基酸是污泥厌氧消化过程的重要中间产物，具有L和D两种构型，互为对映体，其特性与立体构型紧密相关。预处理（如碱处理、热处理等）常用来强化污泥厌氧消化过程，预处理会造成蛋白质水解的氨基酸发生外消旋化，形成L和D两种构型氨基酸，但目前未见厌氧消化过程中氨基酸对映体的产生和分布规律及其对厌氧消化影响的报道。本项目的研究内容主要包括蛋白质水解过程中氨基酸及其对映体的产生和分布规律探究，并进一步考察氨基酸及其对映体对厌氧消化过程的影响和作用机制；通过对厌氧消化各阶段的研究，识别其影响的关键过程和影响机理；从厌氧消化关键酶活性、关键基因的转录表达、底物特性变化以及微生物菌群结构的角度，研究其影响和作用机制。研究成果有利于深入理解氨基酸及其对映体在厌氧消化过程中的作用，为调控厌氧消化提供新的角度和方法。

污泥中蛋白质占总有机质比例较高，因此氨基酸是其厌氧消化过程的重要中间代谢产物。氨基酸对映体具有重要的生化功能，且与其立体构型（L型和D型）息息相关。本研究首先分析了污泥厌氧消化过程中氨基酸对映体的产生和分布规律，并将其与甲烷产量进行相关性分析。研究表明碱处理导致污泥中L型和D型氨基酸种类和浓度均有提升，其中半胱氨酸含量最高，苏氨酸的外消旋化率（D/(D+L)）最高(54.0%)，其次为半胱氨酸(45.5%)和丙氨酸(44.0%)，且随着碱处理pH增加而增加。在碱处理pH为11时产甲烷量达到最高，为788.5 mL/g-VS。D-亮氨酸、D-天冬氨酸、L-苏氨酸和L-半胱氨酸与产甲烷之间有强相关关系，其Spearman系数分别为0.894、0.9、0.9和0.9（p<0.05）。.基于上述研究，筛选了四种典型氨基酸对映体，研究其对污泥厌氧消化各个阶段和产甲烷量的影响。研究结果表明，添加同种氨基酸的L型和D型对有机物溶出影响区别不大，但对甲烷产量和辅酶F420的促进作用中L型优于D型。添加L-半胱氨酸组甲烷产量最高，其次为L-丙氨酸和L-天冬氨酸；同时D、L-天冬氨酸、L-半胱氨酸和L-丙氨酸的辅酶F420含量较高。添加L-半胱氨酸有助于Clostridia和Methanobacterium的相对丰度增加，以耗氢产甲烷为主。研究进一步探究了D、L-半胱氨酸对厌氧消化过程影响及作用机制。当D、L-半胱氨酸添加浓度分别为150和50 mg/L时，SCOD降解率最高，累计甲烷产量亦达到最大值，分别为877.6和906.3 mL/g-VS。而L-半胱氨酸添加浓度为200 mg/L时，甲烷产量将受到抑制。在溶解和水解阶段，半胱氨酸L型促进效果优于D型。在酸化阶段中，L-半胱氨酸实验组的乙酸和ACK酶活含量最高，而D/L-半胱氨酸对同型产乙酸作用不明显。在产甲烷阶段中，L型半胱氨酸对耗氢产甲烷过程促进作用优于D型。由此可见，添加氨基酸可为调控污泥厌氧消化产甲烷提供新思路。