Metagenomic approach reveals the fate of antibiotic resistance genes in a temperature-raising anaerobic digester treating municipal sewage sludge

The fate of diverse antibiotic resistance genes (ARGs) in anaerobic digestion (AD) system under different temperature remains largely unknow. This study employed the shotgun metagenomic sequencing and 16S rRNA amplicon sequencing approaches and investigated the shifts of composition and abundance of more than 1360 ARGs, as well as microbial community, in a temperature-raising AD system (from 35 °C to 55 °C) treating municipal sewage sludge. Results suggested that the six ARGs, inlcuding macB, tetA(58), bcrA, evgS, mtrA, and oleC, were predominated in digested sludge. More than 407ARG subtypes (30%) could be largely reduced under high temperature condition. Thermophilic AD (55 °C) provided a better removal for tetracycline, macrolide, penam, fluoroquinolone, acridine dye, and peptide resistance genes when compared to mesophilic AD (35 °C). The removal rates of these genes reached 29.7%–32.3%. In addition, at the end of thermophilic digestion process, the relative abundance of Firmicutes increased (from 18.22% to 74.89%), while Proteobacteria and Chloroflexi both decreased, from 36.39% to 6.80% and 20.29%–2.64%. This study further proposed the underlying mechanisms of effective elimination of ARGs under high temperature by: (1) the reduction of genes encoding antibiotic efflux pump, (2) the promotion of sludge biomass degradation rate, (3) the loss of microbial community diversity in AD systems, and (4) the inactivation of multidrug-resistant bacteria. The significance of current study is in characterizing the fate of ARGs across AD process, which allows to correlate diverse genetic properties with digesters’ operational condition. Results provided an insight into the manipulation of AD technology (e.g. temperature) to efficiently remove ARGs from municipal sewage sludge.