Effects of Pyroligneous Acid on Diversity and Dynamics of Antibiotic Resistance Genes in Alfalfa Silage

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. IMPORTANCE Antibiotic resistance genes (ARGs) are considered environmental pollutants posing a potential human health risk. Silage is an important and traditional feed, mainly for ruminants. ARGs in silages might influence the diversity and distribution of ARGs in animal intestinal and feces and then the manure and the manured soil. However, the diversity and dynamics of ARGs in silage during fermentation are still unknown. We ensiled alfalfa, one of the most widely used forages, with or without pyroligneous acid (PA), which was proved to have the ability to reduce ARGs in soils. The results showed that ARGs in alfalfa silage were abundant and were influenced by the fermentation parameters and microbial community. The majority of ARGs in alfalfa silage reduced during fermentation. The addition of PA could improve silage quality and reduce ARG pollution in alfalfa silage. This study can provide useful information for understanding and controlling ARG pollution in animal production.