Digestate from Agricultural Biogas Plants as a Reservoir of Antimicrobials and Antibiotic Resistance Genes-Implications for the Environment

Abstract

Antimicrobials and antibiotic resistance genes (ARGs) in substrates processed during anaerobic digestion in agricultural biogas plants (BPs) can reach the digestate (_D), which is used as fertilizer. Antimicrobials and ARGs can be transferred to agricultural land, which increases their concentrations in the environment. The concentrations of 13 antibiotics in digestate samples from biogas plants (BPs) were investigated in this study. The abundance of ARGs encoding resistance to beta-lactams, tetracyclines, sulfonamides, fluoroquinolones, macrolide-lincosamide-streptogramin antibiotics, and the integrase genes were determined in the analyzed samples. The presence of cadmium, lead, nickel, chromium, zinc, and mercury was also examined. Antimicrobials were not eliminated during anaerobic digestion. Their concentrations differed in digestates obtained from different substrates and in liquid and solid fractions (ranging from 62.8 ng/g clarithromycin in the solid fraction of sewage sludge digestate to 1555.9 ng/L doxycycline in the liquid fraction of cattle manure digestate). Digestates obtained from plant-based substrates were characterized by high concentrations of ARGs (ranging from 5.73 × 10² copies/g_DcfxA to 2.98 × 10⁹ copies/g_Dsul1). The samples also contained mercury (0.5 mg/kg dry mass (dm)) and zinc (830 mg/kg dm). The results confirmed that digestate is a reservoir of ARGs (5.73 × 10² to 8.89 × 10¹⁰ copies/g_D) and heavy metals (HMs). In addition, high concentrations of integrase genes (10⁵ to 10⁷ copies/g_D) in the samples indicate that mobile genetic elements may be involved in the spread of antibiotic resistance. The study suggested that the risk of soil contamination with antibiotics, HMs, and ARGs is high in farms where digestate is used as fertilizer.

Keywords: ARGs; cattle manure; heavy metals; maize silage; sewage sludge; slaughterhouse waste.

Figure 1

Concentrations of antibiotics in (a) liquid and (b) solid fractions of digestate. Error bars denote standard deviation (SD).

Figure 1

Concentrations of antibiotics in (a) liquid and (b) solid fractions of digestate. Error bars denote standard deviation (SD).

Figure 2

Normalized heatmap (%) presenting the absolute abundance (AA) of antibiotic resistance and integrase genes in digestate samples (number of copies/g of digestate).

Figure 3

Principal Component Analysis (PCA) based on the distribution of ARGs and integrase genes, pharmaceuticals, and heavy metals (HMs) in all investigated samples. Mean loadings for ARGs and the content of antibiotics and HMs, and mean PC scores for biogas plants: A—biogas plant 1 (BP1), B—biogas plant 2 (BP2) and C—biogas plant 3 (BP3).