Removal of Diverse and Abundant ARGs by MF-NF Process from Pig Manure and Digestate

Abstract

Antimicrobial resistances are emerging as one main threat to worldwide human health and are expected to kill 10 million people by 2050. Intensive livestock husbandry, along with biogas digestate, are considered as one of the biggest ARG reservoirs. Despite major concerns, little information is available on the diversity and abundance of various ARGs in small to large scale pig farms and biogas digestate slurry in Germany, followed by their consequent removal using microfiltration (MF)-nanofiltration (NF) process. Here, we report the identification and quantification of 189 ARGs in raw manure and digestate samples, out of which 66 ARGs were shared among manures and 53 ARGs were shared among both manure and digestate samples. The highest reported total ARG copy numbers in a single manure sampling site was 1.15 × 10⁸ copies/100 µL. In addition, we found the absolute concentrations of 37 ARGs were above 10⁵ copies/100 μL. Filtration results showed that the highly concentrated ARGs (except aminoglycoside resistance ARGs) in feed presented high log retention value (LRV) from 3 to as high as 5 after the MF-NF process. Additionally, LRV below 2 was noticed where the initial absolute ARG concentrations were ≤10³ copies/100 μL. Therefore, ARG removal was found to be directly proportional to its initial concentration in the raw manure and in digestate samples. Consequently, some ARGs (tetH, strB) can still be found within the permeate of NF with up to 10⁴ copies/100 μL.

Keywords: antibiotic resistance genes; antimicrobial resistance; digestate; manure; microfiltration; nanofiltration.

Figure 1

Comparison of (A) total detected ARGs and (B) log value of ARG copy numbers among three sampling sites, conferring resistance to different antibiotics, where GC referred to gene copy numbers.

Figure 2

Absolute ARG concentration (per 100 µL) profile, resistance to different antibiotic groups in each sampling site. Zones (A), (B), and (D) are enriched in all sampling sites; (C), (E), and (F) are enriched in sites 1 and 2 but not in site 3; Zones I, II, and III denoted the absolute ARG copy numbers ≤ 10³ per 100 µL.

Figure 3

Log retention values (LRVs) of individual ARGs of pig manure and digestate samples of sampling sites 1, 2, and 3. Zones (A), (B), and (D) were enriched in raw samples of all the sampling sites; (C), (E), and (F) were enriched in the raw samples of sites 1 and 2 but not in the raw digestate sample of site 3; Zones I, II, and III denoted the absolute ARG copy numbers ≤10³/100 µL in the raw samples of all sampling sites.