空间异质性对猪粪高固体厌氧消化中抗生素抗性基因的影响机制

The proliferation of antibiotic resistance genes (ARGs) in soil and crops is a problem to be solved for agricultural resources ultilization of swine manure. High solid anaerobic digestion (HSAD) is one of the important measures to control the proliferation of ARGs from swine manure; and spatial heterogeneity of HSAD is the main factor influencing the stability of anaerobic digestion system, affecting the fate of ARGs. However, it does not clarify the influence mechanism of spatial heterogeneity of HSAD on ARGs. So, this study will plan to construct two HSAD systems, static and dynamic HSAD. From three dimensions of vertical, horizontal and time during anaerobic digestion system, the spatial heterogeneity of anaerobic digestion substrate will be analyzed; high-throughput sequencing and high-throughput quantitative PCR will be used to study the temporal and spatial differences of microbial community, mobile genetic elements and ARGs in HSAD; the main driving factors influencing the fate of ARGs will be analyzed, and the changes of the main driving factors will be explored with spatial heterogeneity, then the response mechanism of the fate of ARGs to microbial community, environmental factors and so on will be analyzed from the perspective of spatial heterogeneity. The results will provide theoretical reference and scientific guidance for the control on the proliferation of ARGs by HSAD.

抗生素抗性基因（Antibiotic Resistance Genes，ARGs）在土壤和作物中扩散是猪粪农业资源化利用亟待解决的问题。高固体厌氧消化（High Solid Anaerobic Digestion，HSAD）是控制猪粪中ARGs扩散的重要措施，其空间异质性是影响消化体系稳定性的重要因素，并影响ARGs归趋。然而，HSAD空间异质性对ARGs的影响机制尚未明确。因此，本研究拟构建HSAD静态和动态两个体系，从消化体系纵横时间三个维度分析消化底物空间异质性；采用高通量测序和高通量定量PCR技术研究HSAD中微生物群落、可移动遗传元件、ARGs的时空差异，解析影响ARGs归趋的主要驱动因子，探索随空间异质性ARGs主要驱动因子的变化，并从空间异质性角度阐释ARGs演化对微生物群落和环境因子等的响应机制。研究结果将为猪粪HSAD控制ARGs扩散提供理论参考和科学指导。

猪粪和秸秆高固体厌氧消化过程中底物存在显著的空间异质性，对微生物和抗生素抗性基因产生显著的影响。本研究建立了猪粪和秸秆高固体厌氧消化系统，每日最高产甲烷量为7.46 ml·g-1VS·d-1，140天累积产甲烷量是400.17 ml·g-1VS，厌氧消化底物在厌氧消化初期出现膨胀，膨胀高度达到底物初始高度的62.89%。厌氧消化过程中环境因子pH值、EC、TS、VS、VFAs、铵态氮、游离铵在消化罐中发生明显的空间变化：以纵向为主，即从上到下，pH值纵向变化不大，EC值从上到下逐渐增大，TS、VS和VFAs从上到下逐渐减小，铵态氮在厌氧消化前期上层含量高，后期下层含量高。采用荧光定量 PCR 技术对微生物进行16S rDNA 绝对定量，16S rDNA高通量测序技术测定微生物的多样性。16S rDNA的绝对拷贝数，在消化罐中从上到下显著降低；产甲烷菌主要是甲烷八叠球菌，该菌在消化罐上层和下层的相对丰度差异较小，但是绝对含量上层显著高于下层；分解秸秆的微生物在上层中相对丰度显著高于下层，下层中分解易降解物质的微生物相对丰度显著高于上层。通过PCA、RDA和VPA等分析，表明产甲烷菌群落变化主要受到微生物群落和环境因子的共同影响，微生物群落和环境因子存在明显的空间差异性。项目资助发表SCI一区论文2篇，待发表论文2篇。后续研究结果，将进一步完善高固体厌氧消化过程中底物空间异质性对抗生素抗性基因的影响。