厌氧消化过程中污泥和猪粪源抗生素抗性基因转归的底物响应机制研究

Sewage sludge and swine manure are important reservoirs of antibiotic resistance genes (ARGs) in the environment, and anaerobic digestion is an important way of resource utilization of sewage sludge and swine manure. The investigation of the effects of characteristics of substrate on the fate of ARGs during anaerobic digestion was limited, and the existed little information distinguish extracellular ARGs (eARGs) from intracellular ARGs (iARGs). The project aims to systematically elaborate the mechanisms and the fate of ARGs response to the substrate during anaerobic digestion, in which sewage sludge and swine manure are targeted. The fate of ARGs response to the substrate type is investigated through the comparative analysis of the fate of iARGs and eARGs between sewage sludge and swine manure during anaerobic digestion concerning the fermentation, acidification and methanogenesis phase, and the expression of ARGs is also involved. The critical factors concerning the manure and sludge borne microbial community composition, ARGs composition and organics composition are revealed during anaerobic digestion of sewage sludge and swine manure. Meanwhile, the mechanisms concerning vertical gene transfer, horizontal gene transfer and the occurrence in the extracellular are clarified to reveal the dynamic balance between iARGs and eARGs, and then, the mechanisms of the response of the fate of ARGs to the substrate during anaerobic digestion are uncovered. The predicted achievements could provide the theoretical basis for the ARGs control through the anaerobic digestion of sewage sludge and swine manure.

现阶段针对厌氧消化过程中抗生素抗性基因（antibiotic resistance gene，ARGs）的转归，意见不一，底物性质的影响尚不清楚，细胞内、外ARGs的转归差异认识不足。本项目以污泥和猪粪源ARGs为研究对象，解析厌氧消化过程中ARGs转归的底物响应机制；通过对比分析污泥和猪粪厌氧消化不同阶段（水解、产酸和产甲烷阶段）胞内、外ARGs的赋存、转归和表达特征，明确底物类型对厌氧消化过程中ARGs转归的影响；通过揭示污泥和猪粪源微生物、本底ARGs组成及有机物组成对厌氧消化过程中ARGs转归的影响，明确厌氧消化过程中影响ARGs转归的底物组成关键因子；通过解析胞内、外ARGs的垂直转移、水平转移和胞外赋存机制，揭示底物类型和组分对胞内、外ARGs转归动态平衡的影响，阐明厌氧消化过程中ARGs转归的底物响应机制，为通过厌氧消化技术削减污泥和猪粪源ARGs提供理论依据和技术支撑。

本项目以环境中抗生素抗性基因（Antibiotic resistance genes, ARGs）典型的源（污泥和畜禽粪便）为研究对象，解析厌氧消化过程中ARGs 转归的底物响应特征，并明确其分子机制。首先，宏基因组学解析结果表明，猪粪（Pig manure, PM）和鸡粪（Chicken manure, CM）中均以氨基糖苷类（aminoglycoside，41.7% vs 26.2%），四环素类（tetracycline，27.7% vs 21.6%）和MLS（24.2% vs 21.4%）的ARGs为主，并且在CM中含有较高丰度的氯霉素类（chloramphenicol，9.6%）ARGs。而污泥（Sewage sludge, SS）中ARGs的赋存显著区别于PM和CM，并以磺胺类（sulfonamide）ARGs（24.9%）为主，其次为氨基糖苷类（18.4%）和多重耐药性ARGs（16.5%）。其次，厌氧消化过程中，底物类型对胞内ARGs（intracellular ARGs, iARGs）和胞外ARGs（extracellular, eARGs）的归趋均具有显著影响（p＜0.05），PM, CM和SS中eARGs在总ARGs所占比列分别为5.47%，0.29%和0.04%。底物本地微生物影响有限，底物有机组分的差异是决定不同底物厌氧消化中ARGs归趋的主要因素。不同底物中抗生素含量差异较大，并表现出畜禽粪便远高于SS，但在不同底物厌氧消化过程中，ARGs的归趋同底物中抗生素浓度变化无显著相关性，且厌氧消化对抗生素具有较好的去除效果（＞90%）。普鲁克分析（Procrustes analysis）和结构方程模型（Structural equation models, SEM）表明，微生物群落结构的演替是影响iARGs归趋的主要因素，而eARGs的归趋与微生物群落结构的变化无显著相关性。总之，不同底物中ARGs归趋的影响是由其厌氧体系中微生物群落结构的变化所决定，不同底物的有机组分决定了厌氧消化工艺所需微生物群落结构和功能。作为ARGs的宿主菌系，厌氧消化相关功能微生物群落在不同有机组分的竞争利用中，形成了微生物群落结构的演替，从而决定了ARGs的归趋。