死菌DNA对厌氧消化污泥中抗生素抗性基因及微生物群落分析的干扰

Interference of Dead Cell DNA on the Analysis of Antibiotic Resistance Genes and Microbial Community in Anaerobic Digestion Sludge

为评估死菌DNA对厌氧消化污泥抗生素抗性基因(ARGs)和微生物群落分析的潜在干扰,本研究对3种不同类型厌氧消化污泥进行叠氮溴化丙锭(PMA)处理,比较在PMA屏蔽死菌DNA PCR扩增情况下污泥ARGs和微生物群落分析结果与未经PMA处理情况下的差异.结果表明,经PMA处理后,剩余污泥自厌氧消化样品和高含固厌氧消化污泥样品中的ARGs丰度分别下降了41%~86%和74%~98%;污泥水解液厌氧消化15 d后的污泥样品中ARGs下降幅度相对较小,但降幅最高也达到34%.PMA处理对3个来源不同的厌氧消化污泥微生物群落组成分析结果呈现不同程度的影响,对高含固厌氧消化污泥的微生物群落结构分析影响最为显著.在经PMA处理与未经PMA处理两种情况下,厌氧消化污泥ARGs与微生物群落组成相关性分析的结果也截然不同.研究证明了死菌DNA对厌氧消化污泥ARGs和微生物群落分析的潜在干扰,采用PMA预处理能够更准确地反映厌氧消化污泥中的微生物群落及菌体携带ARGs的特征.

To investigate the potential interference of dead bacteria DNA on the analysis of antibiotic resistance genes(ARGs)and microbial communities in anaerobic digestion sludge,three different types of anaerobic digestion sludge were treated with propidium monoazide(PMA)in this study,and the results of subsequent ARGs and microbial community analysis with the interference of dead cell DNA blocked by PMA was compared to that without PMA treatment.It was found that after PMA treatment,the absolute abundance of the ARGs in the anaerobic digestion sludge from self-anaerobic digestion system of excess sludge and from high solid sludge anaerobic digestion system decreased by 41%-86%,and 74%-98%,respectively.ARGs abundance in the sludge from the anaerobic digestion system with sludge hydrolyzate as a substrate and anaerobic granular sludge as inoculum after 15 days of digestion considerably decreased with PMA treatment.However,its ARGs abundance still declined by up to 34%.PMA treatment influenced the analysis of microbial community of the three types of anaerobic digestion sludge to different degrees,in which the influence was the highest on the community structure analysis of the sludge from high solid anaerobic digestion system.The results of the correlation analysis between the ARGs abundance and the microbial community composition were completely different in the case with PMA treatment and without PMA treatment.This study proved the potential interference of dead cell DNA on the analysis of ARGs and microbial community in anaerobic digestion sludge.PMA pretreatment could achieve a more accurate analysis of the microbial community and ARGs characteristics in anaerobic digestion sludge.