摘要
      抗生素耐药性基因（ARGs）因其对人类和环境的潜在风险而被认为是污染物。本研究的目的是通过宏基因组分析，研究生物炭生产中的废物焦木酸（PA）对苜蓿青贮过程中ARGs的发酵特性、多样性和动力学的影响。结果表明PA降低（P < 0.05）干物质损失、pH值、产气量、大肠杆菌数、蛋白酶活性、非蛋白-N、氨-N和丁酸含量增加（P < 0.05）乳酸含量。在发酵过程中，细菌、厚壁菌门和乳杆菌分别在王国、门和属水平上最丰富。热解酸降低了细菌和厚壁菌门的相对丰度，增加了乳杆菌的相对丰度。检测到的ARGs属于36个药物类别，主要包括大环内酯类、四环素类、林可酰胺类和氯霉素类。这些类型的ARGs在发酵过程中减少，并被PA进一步减少。这些类型的ARGs呈正相关（P < 0.05）与发酵参数如pH值和氨氮含量以及与细菌群落的关系。在属水平上，前几类药物，包括大环内酯、四环素、林可酰胺、氯霉素、恶唑烷酮、链菌素、胸膜实用素和糖肽，与ARGs的潜在宿主葡萄球菌、链球菌、李斯特菌、芽孢杆菌、克雷伯菌、梭菌和肠杆菌呈正相关。总体而言，苜蓿青贮饲料中的ARGs含量丰富，受发酵参数和微生物群落组成的影响。围堵可能是一种可行的方法来减少牧草中的ARGs。添加PA不仅可以提高苜蓿青贮饲料的发酵质量，而且可以减少ARG污染。
ABSTRACT
Antibiotic resistance genes (ARGs) are recognized as contaminants due to their potential risk for human and environment. The aim of the present study is to investigate the effects of pyroligneous acid (PA), a waste of biochar production, on fermentation characteristics, diversity, and dynamics of ARGs during ensiling of alfalfa using metagenomic analysis. The results indicated that PA decreased (P < 0.05) dry matter loss, pH value, gas production, coliform bacteria count, protease activity, and nonprotein-N, ammonia-N, and butyric acid contents and increased (P < 0.05) lactic acid content during ensiling. During fermentation, Bacteria, Firmicutes, and Lactobacillus were the most abundant at kingdom, phylum, and genus levels, respectively. Pyroligneous acid reduced the relative abundance of Bacteria and Firmicutes and increased that of Lactobacillus. The detected ARGs belonged to 36 drug classes, including mainly macrolides, tetracycline, lincosamides, and phenicol. These types of ARGs decreased during fermentation and were further reduced by PA. These types of ARGs were positively correlated (P < 0.05) with fermentation parameters like pH value and ammonia-N content and with bacterial communities. At the genus level, the top several drug classes, including macrolide, tetracycline, lincosamide, phenicol, oxazolidinone, streptogramin, pleuromutilin, and glycopeptide, were positively correlated with Staphylococcus, Streptococcus, Listeria, Bacillus, Klebsiella, Clostridium, and Enterobacter, the potential hosts of ARGs. Overall, ARGs in alfalfa silage were abundant and were influenced by the fermentation parameters and microbial community composition. Ensiling could be a feasible way to mitigate ARGs in forages. The addition of PA could not only improve fermentation quality but also reduce ARG pollution of alfalfa silage.

https://journals.asm.org/doi/full/10.1128/spectrum.01554-22